U.S. patent application number 15/781370 was filed with the patent office on 2018-11-08 for open-top microfluidic device with structural anchors.
The applicant listed for this patent is EMULATE, INC.. Invention is credited to Geraldine Hamilton, Christopher David Hinojosa, Katia Karalis, S. Jordan Kerns, Daniel Levner, Carol Lucchesi, Justin Nguyen, Antonio Varone, Norman Wen, Lina Williamson.
Application Number | 20180320125 15/781370 |
Document ID | / |
Family ID | 58798085 |
Filed Date | 2018-11-08 |
United States Patent
Application |
20180320125 |
Kind Code |
A1 |
Levner; Daniel ; et
al. |
November 8, 2018 |
OPEN-TOP MICROFLUIDIC DEVICE WITH STRUCTURAL ANCHORS
Abstract
A microfluidic device is contemplated comprising an open-top
cavity with structural anchors on the vertical wall surfaces that
serve to prevent gel shrinkage-induced delamination, a porous
membrane (optionally stretchable) positioned in the middle over a
microfluidic channel(s). The device is particularly suited to the
growth of cells mimicking dermal layers.
Inventors: |
Levner; Daniel; (Brookline,
MA) ; Hinojosa; Christopher David; (Cambridge,
MA) ; Wen; Norman; (West Roxbury, MA) ;
Varone; Antonio; (Newton, MA) ; Nguyen; Justin;
(Medford, MA) ; Williamson; Lina; (Chape Hill,
NC) ; Kerns; S. Jordan; (Reading, MA) ;
Karalis; Katia; (Brookline, MA) ; Hamilton;
Geraldine; (Cambridge, MA) ; Lucchesi; Carol;
(Westwood, MA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
EMULATE, INC. |
Boston |
MA |
US |
|
|
Family ID: |
58798085 |
Appl. No.: |
15/781370 |
Filed: |
December 2, 2016 |
PCT Filed: |
December 2, 2016 |
PCT NO: |
PCT/US16/64814 |
371 Date: |
June 4, 2018 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
62263492 |
Dec 4, 2015 |
|
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|
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12M 23/16 20130101;
C12M 25/02 20130101; C12N 5/0698 20130101; C12N 2501/165 20130101;
C12M 35/08 20130101; C12N 5/0656 20130101; C12N 5/069 20130101;
G01N 33/54366 20130101; C12M 23/38 20130101; C12M 35/04 20130101;
C12N 5/0688 20130101; C12N 5/0697 20130101; C12N 5/0619 20130101;
C12N 5/0629 20130101; C12N 5/0622 20130101; G01N 33/50 20130101;
G01N 33/5082 20130101 |
International
Class: |
C12M 1/42 20060101
C12M001/42; C12M 1/12 20060101 C12M001/12; C12M 3/06 20060101
C12M003/06; C12N 5/071 20060101 C12N005/071; C12N 5/077 20060101
C12N005/077; C12M 1/00 20060101 C12M001/00; C12N 5/0793 20060101
C12N005/0793; C12N 5/079 20060101 C12N005/079; G01N 33/50 20060101
G01N033/50 |
Claims
1. A device comprising i) a chamber, said chamber comprising a
non-linear lumen and projections in the lumen, said lumen
comprising ii) a gel matrix secured by said projections, said gel
matrix positioned above iii) a porous membrane, said membrane
positioned above one or more iv) fluidic channels.
2. The device of claim 1, wherein fibroblasts are within the gel
matrix and keratinocytes are on top of the gel matrix.
3. The device of claim 2, wherein the keratinocytes comprise more
than one layer on top of the gel matrix.
4. The device of claim 1, wherein a layer of endothelial cells is
positioned on the bottom of the membrane so as to be in contact
with the fluidic channels.
5-9. (canceled)
10. The device of claim 1, wherein said non-linear lumen is
circular.
11. The device of claim 1, further comprising a removable
cover.
12. The device of claim 1, wherein said device is a microfluidic
device and said fluidic channels are microfluidic channels.
13-27. (canceled)
28. A microfluidic device comprising i) a chamber, said chamber
comprising a lumen, said lumen comprising ii) a gel matrix
comprising at least one of neurons and astrocytes, said gel matrix
positioned above iii) a porous membrane, said membrane comprising
brain microvascular endothelial cells in contact with iv)
microfluidic channels.
29. The microfluidic device of claim 28, wherein neurons are on, in
or under the gel matrix.
30. The microfluidic device of claim 28, wherein astrocytes are on,
in or under the gel matrix.
31. A method comprising: a) providing a microfluidic device
comprising i) a chamber, said chamber comprising a non-linear
lumen, said lumen comprising ii) a gel matrix, said gel matrix
positioned above iii) a porous membrane, said membrane positioned
above one or more iv) fluidic channels; and b) stretching said gel
matrix.
32. The method of claim 31, wherein the gel matrix has a thickness
of between 0.5 mm and 3.5 mm.
33. The method of claim 32, wherein said stretching is uniform
across the thickness of the gel.
34. The method of claim 31, wherein said stretching causes the
entire gel matrix to expand.
35. The method of claim 31, wherein said gel matrix comprises
cells.
36. The method of claim 35, wherein said cells comprise lung cells.
Description
FIELD OF THE INVENTION
[0001] A microfluidic device is contemplated comprising an open-top
cavity with structural anchors on the vertical wall surfaces that
serve to prevent gel shrinkage-induced delamination, a porous
membrane (optionally stretchable) positioned in the middle over a
microfluidic channel(s).
BACKGROUND
[0002] In vitro models involving thick gels, i.e. >0.2 mm in
thickness (and more typically >0.5 mm in thickness), have a
common problem of shrinkage due to evaporation of water and
tissue-induced stresses, which cause delamination from surfaces
containing the gel. This issue prevents robust implementation of 3D
gels with microfluidic systems, which can introduce physiologically
relevant shear forces and concentration gradients. In addition,
such delamination will prevent any attempt to apply mechanical
forces, for example stretching or compressing, on the gel
itself.
[0003] 3D gel in vitro models are currently performed in transwells
or well plates, where shrinkage-induced delamination is not
important. Prior art solutions to gel shrinkage include
transplanting the 3D gel post-shrinking, and pre-compressing the
gel, both of which are equivalent to pre-shrinking the gel before
its use. Past microfluidic systems that include mechanical
stretching focused on stretching a thin membrane as opposed to
uniformly stretching a thick (>0.5 mm) gel.
SUMMARY OF THE INVENTION
[0004] A microfluidic device is contemplated comprising an open-top
cavity with structural anchors on the vertical wall surfaces that
serve to prevent gel shrinkage-induced delamination, a porous
membrane (optionally stretchable) positioned in the middle over a
microfluidic channel(s). By preventing delamination, this allows
for implementation of 3D gels with microfluidic systems. The device
is particularly suited to the growth of cells mimicking dermal
layers, allowing for the testing of cosmetics and candidate drugs
(including aerosols).
[0005] In one embodiment, the present invention contemplates making
measurements relating to electrophysiology of cells in an open-top
chip. It is not intended that the present invention be limited by
the type of cells; a variety of excitable cells is contemplated.
Moreover, such measurements can be done whether the open-top chip
comprises a gel or not. In one embodiment, the present invention
contemplates a device, comprising a first structure defining a
first chamber, the first chamber comprising an open top surface
region; a second chamber, wherein a first interface region is
formed between the first chamber and the second chamber ; a
membrane disposed at the first interface region, the membrane
including a first side facing the first chamber and a second side
facing the second chamber; one or more cells disposed in at least
one of the first chamber and the second chamber; and one or more
electrodes. In one embodiment, at least one of the first chamber
and second chamber comprises a fluidic channel. In one embodiment,
at least one of the one or more electrodes is disposed in physical
contact with said membrane. In one embodiment, said performing an
electrophysiological measurement comprises performing a patch-clamp
measurement. In one embodiment, said device further comprises a
cover disposed on top of at least part of the said open top surface
region, and wherein at least one of the one or more electrodes is
disposed in physical contact with the cover. In one embodiment, at
least one of the one or more electrodes is present within the first
chamber and crosses the open top surface region. While not
intending to be limited to the cell type, in one embodiment, said
cells comprise at least one of neurons and astrocytes. In another
embodiment, said cells comprise at least one of retinal rods and
retinal cones. In one embodiment, said cells comprise at least one
of skeletal muscle, smooth muscle and cardiac muscle.
[0006] The present invention also contemplates, in one embodiment,
a layered structure comprising i) fluidic channels covered by ii) a
porous membrane, said membrane comprising iii) a layer of cells and
said membrane positioned below iv) a gel matrix. In one embodiment,
there is a removable cover over the gel matrix (and/or cells).
While not intending to be limited to any particular cell type, in
one embodiment, the cells are brain microvascular endothelial
cells. In one embodiment of the layered structure, it further
comprises neurons on, in or under the gel matrix. In still another
embodiment of the layered structure, it further comprises
astrocytes on, in or under the gel matrix. Cells can be positioned
in various different places (in or on the layered structure). In
one embodiment, the layer of brain microvascular endothelial cells
is positioned on the bottom of the membrane so as to be in contact
with the fluidic channels. It is not intended that the present
invention be limited to any particular source of cells. In one
embodiment, the brain microvascular endothelial cells are primary
cells.
[0007] It is not intended that the present invention be limited to
embodiments with only one gel or gel layer. In one embodiment, the
layered structure further comprises a second gel matrix (e.g.
positioned under said membrane).
[0008] The gel(s) or coatings can be patterned or not patterned.
Moreover, when patterned, the pattern need not extend to the entire
surface. For example, in one embodiment, at least a portion of said
gel matrix is patterned.
[0009] To make measurements, electrodes can be included in the
layered structure, i.e. electrodes configured for measuring the
electrophysiology of cells, such as brain microvascular endothelial
cells. Other cells can also be tested (e.g. muscle cells).
[0010] It is not intended that the present invention be limited by
the nature or components of the gel matrix or gel coating. In one
embodiment, gel matrix comprises collagen. A variety of thickness
is contemplated. In one embodiment of the layered structure, said
gel matrix is between 0.2 and 6 mm in thickness.
[0011] In yet another embodiment, the present invention
contemplates a microfluidic device comprising i) a chamber, said
chamber comprising a lumen and projections in the lumen, said lumen
comprising ii) a gel matrix anchored by said projections, said gel
matrix positioned above iii) a porous membrane, said membrane in
contact with iv) fluidic channels. In one embodiment, said membrane
comprises cells. The projections serve as anchors for the gel. The
projections, in one embodiment, project outward from the side
walls. The projections, in another embodiment, project upward. The
projects, in another embodiment, project downward. The projections
can take a number of forms (e.g. a T structure, a Y structure, a
structure with straight or curving edges, etc.). In some
embodiments, there are two or more projections; in other
embodiments, there are four or more projections to anchor the gel
matrix. In one embodiment, the membrane is above said fluidic
channels. In one embodiment the cells comprise a layer of brain
microvascular endothelial cells (BMECs). In one embodiment, the
BMECs are positioned on the bottom of the membrane so as to be in
contact with the fluidic channels. In one embodiment, it further
comprises neurons on, in or under the gel matrix. In one
embodiment, it further comprises a second gel matrix (e.g.
positioned under said membrane). While not limited to the nature or
source of the cells, in one embodiment, the brain microvascular
endothelial cells are primary cells. In one embodiment, it further
comprises pericytes on, in or under the gel matrix. In one
embodiment, said gel matrix is under a removable cover. In one
embodiment, said gel matrix is patterned (or at least a portion of
it is patterned). In one embodiment, the device further comprises
electrodes, e.g. electrodes are configured for measuring the
electrophysiology of said brain microvascular endothelial cells. In
one embodiment, said gel matrix comprises collagen. In one
embodiment, said collagen matrix is between 0.2 and 6 mm in
thickness.
[0012] The present invention also contemplates, in one embodiment,
a method of testing, comprising 1) providing a layered structure
comprising i0 fluidic channels covered by ii) a porous membrane,
said membrane comprising iii) a layer of cells in contact with said
fluidic channels, said membrane positioned below iv) a gel matrix,
said gel matrix under a removable cover; and 2) measuring the
electrophysiology of said cells. A variety of cell types can be
tested. In one embodiment, the cells are brain microvascular
endothelial cells. In another embodiment, the cells are muscle
cells. In one embodiment of this method, the layered structure
further comprises v) electrodes configured for measuring the
electrophysiology of said brain microvascular endothelial cells. In
one embodiment, said measuring comprises TEER measurements with
said electrodes. In one embodiment, said TEER measurements indicate
tight cell-to-cell junctions between said brain microvascular
endothelial cells. In another embodiment, said measuring of step 2)
comprises patch clamp measurements, extracellular electrophysiology
measurements, imaging using calcium-sensitive dyes or proteins, or
imaging using voltage-sensitive dyes or proteins. In one
embodiment, said brain microvascular endothelial cells express the
marker Glut 1. In one embodiment of this method, said layered
structure further comprises neurons on, in or under said gel
matrix. In one embodiment, the layered structure further comprises
a second gel matrix positioned under said membrane.
[0013] The present invention contemplates a variety of uses for
these devices and methods. In one embodiment, the present invention
contemplates a method of topically testing an agent (whether a
drug, food, gas, or other substance) comprising 1) providing a) an
agent and b) microfluidic device comprising i) a chamber, said
chamber comprising a lumen and projections into the lumen, said
lumen comprising ii) a gel matrix anchored by said projections and
comprising cell in, on or under said gel matrix, said gel matrix
positioned above iii) a porous membrane and under iv) a removable
cover, said membrane comprising brain microvascular endothelial
cells in contact with v) fluidic channels; 2) removing said
removable cover; and 3) topically contacting said cells in, on or
under said gel matrix with said agent. In one embodiment, said
agent is in an aerosol. In one embodiment, agent is in a liquid,
gas, gel, semi-solid, solid, or particulate form.
[0014] The present invention also contemplates a skin model in the
form of a microfluidic device or layered structure. In one
embodiment, the present invention contemplates a device or layered
structure comprising i) fluidic channels covered by ii) a porous
membrane, said membrane comprising iii) a layer of endothelial
cells and said membrane positioned below iv) a gel matrix
comprising fibroblasts and keratinocytes. In one embodiment, the
gel matrix (and or cells) is covered by a removable cover. In one
embodiment, the fibroblasts are within the gel matrix and the
keratinocytes are on top of the gel matrix. In one embodiment, the
keratinocytes comprise more than one layer on top of the gel
matrix.
[0015] In one embodiment, the present invention contemplates a
microfluidic device comprising i) a chamber, said chamber
comprising a lumen and projections into the lumen, said lumen
comprising ii) a gel matrix anchored by said projections, said gel
matrix comprising fibroblasts and keratinocytes, said gel matrix
positioned above iii) a porous membrane, said membrane comprising
endothelial cells in contact with iv) fluidic channels. In one
embodiment, the gel matrix (and/or cells) is covered by a removable
cover. In one embodiment, the membrane is above said fluidic
channels and wherein the layer of endothelial cells is positioned
on the bottom of the membrane so as to be in contact with the
fluidic channels. In one embodiment, the fibroblasts are within the
gel matrix and the keratinocytes are on top of the gel matrix.
[0016] The present invention contemplates, in one embodiment, a
method of treating endothelial cells, comprising 1) providing a) an
angiogenic or arteriogenic growth factor in solution, b) a layered
structure comprising i) fluidic channels covered by ii) a porous
membrane, said membrane comprising iii) a layer of endothelial
cells in contact with said fluidic channels, said membrane position
below iv) a gel matrix comprising fibroblasts and keratinocytes;
and 2) introducing said solution into said fluidic channels
comprising said angiogenic or arteriogenic growth factor so as to
treat said endothelial cells. In one embodiment, the gel matrix
(and/or cells) is covered by a removable cover. In one embodiment,
prior to said introducing of step 2), the cover is removed.
[0017] The present invention also contemplates, in one embodiment,
a method of testing a drug or other agent on keratinocytes,
comprising 1) providing a) a candidate drug and b) microfluidic
device comprising i) a chamber, said chamber comprising a lumen and
projections into the lumen, said lumen comprising ii) a gel matrix
anchored by said projections, said gel matrix comprising
fibroblasts and keratinocytes, said gel matrix positioned above
iii) a porous membrane, said membrane comprising endothelial cells
in contact with iv) fluidic channels; and 2) contacting said
keratinocytes with said candidate drug. In one embodiment, the gel
matrix (and or cells) is covered by a removable cover. In one
embodiment, prior to said contacting of step 2), said cover is
removed. In one embodiment, the agent or drug is in the form of an
aerosol.
[0018] The present invention also contemplates, in one embodiment,
microfluidic device for simulating a function of a tissue,
comprising: a first structure defining a first chamber, the first
chamber comprising a gel disposed therein and including an opened
region, said gel comprising intestinal epithelial cells in or on
said gel; a second structure defining a second chamber; and a
membrane located at an interface region between the first chamber
and the second chamber, the membrane including a first side facing
toward the first chamber and a second side facing toward the second
chamber, said second side comprising living endothelial cells. In
one embodiment, the gel has a patterned surface.
[0019] The present invention also contemplates, in one embodiment,
a microfluidic device for simulating a function of a tissue,
comprising: a first structure defining a first chamber, the first
chamber comprising a gel disposed therein and including an opened
region, said gel comprising muscle cells in or on said gel; a
second structure defining a second chamber; and a membrane located
at an interface region between the first chamber and the second
chamber, the membrane including a first side facing toward the
first chamber and a second side facing toward the second chamber.
In one embodiment, the gel has a patterned surface.
[0020] In one embodiment, the present invention contemplates a
device comprising i) a chamber, said chamber comprising a
non-linear lumen, said lumen comprising ii) a gel matrix, said gel
matrix positioned above iii) a porous membrane, said membrane
positioned above one or more iv) fluidic channels. In one
embodiment, the fibroblasts are within the gel matrix and
keratinocytes are on top of the gel matrix. In one embodiment, the
keratinocytes comprise more than one layer on top of the gel
matrix. In one embodiment, the layer of endothelial cells is
positioned on the bottom of the membrane so as to be in contact
with the fluidic channels. In one embodiment, the endothelial cells
are primary cells. In one embodiment, the primary cells are small
vessel human dermal microvascular endothelial cells. In one
embodiment, the primary cells are human umbilical vein endothelial
cells. In one embodiment, the primary cells are bone marrow-derived
endothelial progenitor cells. In one embodiment, the keratinocytes
are epidermal keratinocytes. In one embodiment, the non-linear
lumen is circular. In one embodiment, the device further comprises
a removable cover. In one embodiment, the device is a microfluidic
device and said fluidic channels are microfluidic channels.
[0021] In one embodiment, the present invention contemplates a
microfluidic device comprising i) a chamber, said chamber
comprising a circular lumen, said lumen comprising ii) a gel matrix
comprising fibroblasts and keratinocytes, said gel matrix
positioned above iii) a porous membrane, said membrane comprising
endothelial cells in contact with iv) microfluidic channels. In one
embodiment, the membrane is above said fluidic channels and wherein
the layer of endothelial cells is positioned on the bottom of the
membrane so as to be in contact with the fluidic channels. In one
embodiment, the fibroblasts are within the gel matrix and the
keratinocytes are on top of the gel matrix. In one embodiment, the
keratinocytes comprise more than one layer on top of the gel
matrix. In one embodiment, the endothelial cells are primary cells.
In one embodiment, the primary cells are small vessel human dermal
microvascular endothelial cells. In one embodiment, the primary
cells are human umbilical vein endothelial cells. In one
embodiment, the primary cells are bone marrow-derived endothelial
progenitor cells. In one embodiment, the keratinocytes are
epidermal keratinocytes. In one embodiment, the keratinocytes are
human foreskin keratinocytes. In one embodiment, the matrix
comprises collagen. In one embodiment, the collagen matrix is
between 0.2 and 6 mm in thickness.
[0022] In one embodiment, the present invention contemplates a
method of treating endothelial cells, comprising 1) providing a) an
angiogenic or arteriogenic growth factor in solution, b) a layered
structure comprising i) fluidic channels covered by ii) a porous
membrane, said membrane comprising iii) a layer of endothelial
cells in contact with said fluidic channels, said membrane position
below iv) a gel matrix comprising fibroblasts and keratinocytes;
and 2) introducing said solution into said fluidic channels
comprising said angiogenic or arteriogenic growth factor so as to
treat said endothelial cells. In one embodiment, the gel matrix
comprises collagen. In one embodiment, the collagen matrix is
between 0.2 and 6 mm in thickness.
[0023] In one embodiment, the present invention contemplates a
fluidic cover comprising a fluidic channel, said fluidic cover
configured to engage a microfluidic device. In one embodiment, the
microfluidic device comprises an open chamber, and wherein said
fluidic cover configured to cover and close said open chamber. In
one embodiment, the fluidic cover further comprises one or more
electrodes.
[0024] In one embodiment, the present invention contemplates an
assembly comprising a fluidic cover comprising a fluidic channel,
said fluidic cover detachably engaged with a microfluidic device.
In one embodiment, the microfluidic device comprises an open
chamber, and wherein said fluidic cover configured to cover and
close said open chamber. In one embodiment, the open chamber
comprises a non-linear lumen. In one embodiment, the non-linear
lumen is circular. In one embodiment, the fluidic cover further
comprises one or more electrodes.
[0025] In one embodiment, the present invention contemplates a
method of making an assembly, comprising: a) providing a fluidic
cover comprising a fluidic channel, said fluidic cover configured
to engage b) a microfluidic device, said microfluidic device
comprises an open chamber, and wherein said fluidic cover
configured to cover and close said open chamber; and b) detachably
engaging said microfluidic device with said fluidic cover so as to
make an assembly. In one embodiment, the open chamber comprises a
non-linear lumen. In one embodiment, the non-linear lumen is
circular. In one embodiment, the fluidic cover further comprises
one or more electrodes.
[0026] In one embodiment, the present invention contemplates a
microfluidic device comprising i) a chamber, said chamber
comprising a lumen, said lumen comprising ii) a gel matrix
comprising at least one of neurons and astrocytes, said gel matrix
positioned above iii) a porous membrane, said membrane comprising
brain microvascular endothelial cells in contact with iv)
microfluidic channels. In one embodiment, the neurons are on, in or
under the gel matrix. In one embodiment, the astrocytes are on, in
or under the gel matrix.
DESCRIPTION OF THE FIGURES
[0027] FIG. 1 illustrates an exemplary microfluidic device with a
membrane region having cells thereon according to embodiments of
the present disclosure.
[0028] FIG. 2 is a cross-section of the microfluidic device taken
along line 102-102 of FIG. 1, illustrating the membrane separating
the first microchannel and the second microchannel.
[0029] FIG. 3 illustrates an exploded perspective view of an
exemplary cross-section through an open-top microfluidic device
according to embodiments of the present disclosure.
[0030] FIG. 4 illustrates an exploded perspective view of an
exemplary cross-section through an open-top microfluidic device
with a removable cover according to embodiments of the present
disclosure.
[0031] FIG. 5A illustrates a perspective view of an exemplary
cross-section through an open-top microfluidic device according to
embodiments of the present disclosure.
[0032] FIG. 5B illustrates a perspective view of the exemplary
open-top microfluidic device of FIG. 5A including a gel layer above
a membrane layer in an opened region of a top structure according
to embodiments of the present disclosure.
[0033] FIG. 5C illustrates a perspective view of the exemplary
open-top microfluidic device of FIG. 5B including placement of a
plunger stamp into the opened region of the top structure according
to embodiments of the present disclosure.
[0034] FIG. 5D illustrates a perspective view of the exemplary
open-top microfluidic device of FIG. 5C including a patterned gel
in the opened region of the top structure and a removable cover
disposed above the top structure according to embodiments of the
present disclosure.
[0035] FIG. 5E illustrates a perspective view of the exemplary
open-top microfluidic device of FIG. 5D in an exemplary clamping
device according to embodiments of the present disclosure.
[0036] FIG. 5F illustrates a perspective view of an alternative
exemplary cross-section through an open-top microfluidic device
according to embodiments of the present disclosure.
[0037] FIG. 6 illustrates an exemplary plunger stamp with a
patterned surface according to embodiments of the present
disclosure.
[0038] FIG. 7 illustrates an exemplary pattern for a plunger stamp
according to embodiments of the present disclosure.
[0039] FIG. 8A illustrates a top view of an exemplary stretchable
open-top microfluidic device according to embodiments of the
present disclosure.
[0040] FIG. 8B illustrates a perspective view of the chip top of
the exemplary stretchable open-top microfluidic device of FIG.
8A.
[0041] FIG. 8C illustrates a perspective view of the chip bottom of
the exemplary stretchable open-top microfluidic device of FIG.
8A.
[0042] FIGS. 9 and 10 illustrate exemplary perspective views of
cross-sections through the stretchable open-top microfluidic device
of FIG. 8A.
[0043] FIG. 11 illustrates a partial top view of an exemplary
configuration of multiple parallel channels for a stretchable
open-top micro-fluidic device.
[0044] FIG. 12 illustrates a partial top view of an exemplary
configuration of spiral channel for a stretchable open-top
microfluidic device.
[0045] FIG. 13 shows one plan view embodiment of a device
comprising more than one open-top cavity or chamber, which allows
for direct gel dispensing, cell seeding, and treatments (including
treatment with aerosols and topicals).
[0046] FIG. 14 shows one embodiment of structural anchors along the
cavity/chamber walls in order to prevent shrinkage-induced
delamination of a gel (not shown).
[0047] FIG. 15 shows one embodiment of bottom layer microfluidics,
which allow for shear forces, concentration gradients, and
vascularization (e.g. of endothelial cells).
[0048] FIG. 16 shows one embodiment of vacuum channels designed to
allow for uniform physiological stretching of a thick gel.
[0049] FIG. 17 shows one embodiment of an assembled chip, showing
the open-top chambers above the fluidics.
[0050] FIG. 18 shows the embodiment of FIG. 17, wherein the
membrane is highlighted in order to illustrate the relationship of
the assembled components.
[0051] FIG. 19 shows a vacuum channel cross- section design that
allows bending of the wall about the corner.
[0052] It is not intended that the figures be limiting. The
open-top cavity/chamber can have various geometries other than the
one depicted above: e.g. oval, rectangular slot, ellipse.
Structural anchors can have various geometries other than the one
depicted above. For example, they can have different `head`
geometries and sizes.
[0053] Alternatively, the gel can be maintained with a mesh wall or
micro-pillar array. FIG. 20 shows a top view and elevated side view
of one embodiment of a micro-pillar array. FIG. 21 shows a top view
and elevated side view of one embodiment of a mesh wall or
insert.
[0054] The bottom-layer microfluidics can have various channel
geometries other than the one depicted above, i.e. the channel
height, channel width, and channel path geometry can be changed.
FIG. 22 shows a different design for the microfluidic channels.
[0055] FIGS. 23-26 show various embodiments for microfluidic
devices as contemplated herein that are configured for
electrophysiological measurements (e.g., for example, patch clamp
measurements using transepithelial electric resistance (TEER).
[0056] FIG. 27 illustrates one embodiment of a top view of an
assembled open-top chip microfluidic device of the device depicted
in FIG. 13.
[0057] FIG. 28 illustrates one embodiment of an array of open
chambers in an open-top chip device as contemplated herein.
[0058] FIG. 29 illustrates one embodiment of a stretchable open top
chip device.
[0059] FIG. 29A: A bottom structure with a spiral microchannel with
an inlet well and and outlet well.
[0060] FIG. 29B: A top view of a spiral microchannel configured
with a circular vacuum chamber.
[0061] FIG. 30 illustrates an exploded view of one embodiment of a
stretchable open top chip device demonstrating the layering of a
fluidic top, top structure and bottom structure.
[0062] FIG. 31 illustrates a cut-away view of one embodiment of a
stretchable open top chip device showing the regional placement of
assay cells (e.g., epithelial cells, dermal cells and/or vascular
cells).
[0063] FIG. 32 illustrates a fully assembled view of one embodiment
of a stretchable open top chip device.
[0064] FIGS. 33A and 33B illustrate exploded views of two
embodiments of a stretchable open top chip device.
[0065] FIGS. 34A and 34B illustrate assembled views of a
stretchable open top chip device as depicted in FIGS. 33A and
33B.
[0066] FIGS. 35A and 35B respectively illustrate an assembled
isometric view and an exploded view of a tall channel stretchable
open top chip device.
[0067] FIG. 36 presents a top assembled view of one embodiment of a
stretchable open-top microfluidic chip comprising a fluidic cover
and a single channel.
[0068] FIG. 37 presents a crossectional view of a first embodiment
of a stretchable open top microfluidic chip along plane A of FIG.
36.
[0069] FIG. 37A: Illustrates a fluidic cover in a closed
position.
[0070] FIG. 37B: Illustrates a fluidic cover in an open
position.
[0071] FIG. 38 presents a crossection view of a second embodiment
of a stretchable open top microfludic chip along plane A of FIG.
36.
[0072] FIG. 38A: Illustrates a fluidic cover in a closed
position.
[0073] FIG. 38B: Illustrates a fluidic cover in an open
position.
[0074] FIG. 39 presents an exploded view of the array device
depicted in FIG. 28.
DESCRIPTION OF THE INVENTION
[0075] A microfluidic device is contemplated comprising an open-top
cavity with structural anchors on the vertical wall surfaces that
serve to prevent gel shrinkage-induced delamination, a porous
membrane (optionally stretchable) positioned in the middle over a
microfluidic channel(s). The device can be used in many ways with
many types of tissues and cells. For example, the organ mimic
device described herein can be used for the identification of
markers of disease; assessing efficacy of anti-cancer therapeutics;
testing gene therapy vectors; drug development; screening; and for
studies of particular cells (and arrangements of cells). In one
embodiment, the device serves as a skin model. In this embodiment,
the open-top device provides an uncovered chamber comprising a
skin-like, human or animal tissue that can be tested with drugs,
including topicals and aerosols.
[0076] A. Gel-Containing Skin Model
[0077] In one embodiment, the present invention contemplates a
construct comprising a "dermis" with fibroblasts embedded in a
matrix having a thickness between 0.2 and 6.0 mm, e.g. a collagen I
gel matrix, and an "epidermis", which is comprised of
keratinocytes, e.g. stratified, differentiated keratinocytes. A
matrix such as a collagen gel provides scaffolding, nutrient
delivery, and potential for cell-to-cell interaction. In one
embodiment, the construct further comprises a functional basement
membrane, which separates the epidermis from the dermis.
[0078] In one embodiment, the present invention contemplates a
layered structure comprising i) fluidic channels covered by ii) a
porous membrane, said membrane comprising iii) a layer of
endothelial cells and said membrane position below iv) a gel matrix
comprising fibroblasts and keratinocytes. In a preferred
embodiment, the fibroblasts are within the gel matrix and the
keratinocytes are on top of the gel matrix. In a preferred
embodiment, the keratinocytes comprise more than one layer on top
of the gel matrix. In a preferred embodiment, the layer of
endothelial cells is positioned on the bottom of the membrane and
is in contact with the fluidic channels. In a preferred embodiment,
the fluidic channels provide shear to said endothelial cells.
[0079] It is not intended that the present invention be limited to
the thickness of the gel matrix. However, a preferred range of
thickness is between 0.2 and 6 mm, and more preferably between 0.5
mm and 3.5 mm, and still more preferably approximately 1-2 mm. In a
preferred embodiment, the gel matrix is stretchable. In a preferred
embodiment, the gel matrix is stretched in a manner such that the
entire gel matrix expands, not just a portion of the gel matrix
(such as only the bottom or top of the matrix). In a preferred
embodiment, the gel matrix is stretched by vacuum channels that are
designed to provide pneumatic stretching that is uniform across the
thickness of the gel.
[0080] In a preferred embodiment, the layered structure is
positioned in an open-top microfluidic device (i.e. a device
lacking a top covering), wherein the gel matrix is secured in a
chamber of the device by anchors. In a preferred embodiment, the
surfaces of the device that contact the gel matrix have been
treated to enhance attachment of the gel matrix. In a preferred
embodiment, the surfaces have been plasma treated, i.e. the surface
is activated with ionized gas. It has been found that the surface
treatment, in combination with the anchors, prevent delamination of
the gel from the walls of the chamber.
[0081] In one embodiment, the fluidic channels bring one or more
compounds that will induce the endothelial cells to differentiate.
In one embodiment, the fluidic channels comprise a solution
comprising a vascular endothelial growth factor (VEGF).
[0082] The open-top device provides an uncovered chamber comprising
a skin-like tissue that can be tested with topicals and aerosols.
In one embodiment, drugs are applied topically or transdermally to
the keratinocyte layer(s). As used herein, the term "topical"
refers to administration of an agent or agents (e.g. cosmetic,
medication, vitamin, etc.) on the skin. "Transdermal" refers to the
delivery of an agent (e.g. cosmetic, medication, vitamin, etc.)
through the skin (e.g. so that at least some portion of the
population of molecules reaches underlying layers of the skin).
[0083] In one embodiment, a candidate cosmetic is applied to the
keratinocyte layer(s). As used herein, a "cosmetic" refers to a
substance that aids in the enhancement or protection of the
appearance (e.g. color, texture, look, feel, etc.) or odor of a
subject's skin. A cosmetic may or may not change the underlying
structure of the skin.
[0084] In this skin model, a layer of endothelial cells (ECs) is
positioned on the underside of the membrane facing the fluidic
channels. Endothelial cells and endothelial stem cells will, under
appropriate conditions, migrate and differentiate. In terms of
migration, while not limited to any particular mechanism, it is
believed that this motile process is directionally regulated by
chemotactic, haptotactic, and mechanotactic stimuli and (where
applicable) may require degradation of the extracellular matrix to
enable progression of the migrating cells. It is believed to
involve the activation of several signaling pathways that converge
on cytoskeletal remodeling. Generally, it is been observed that the
endothelial cells extend, contract, and progress forward. In a
preferred embodiment, ECs are grown on a membrane with a porosity
sufficient to allow for this cell migration, i.e. through the
membrane.
[0085] In some embodiments, growth factors or compounds that
enhance the production of the desired cell type(s) can be added to
the perfusion fluid in the fluidic channels. By way of non-limiting
example, erythropoietin stimulates the production of red blood
cells, VEGF stimulates angiogenesis, and thrombopoietin stimulates
the production of megakaryocytes and platelets. "Vascular growth"
is defined here as at least one of vasculogenesis and angiogenesis
and includes formation of one or more of the following:
capillaries, arteries, veins or lymphatic vessels. Blood vessel
formation de novo (vasculogenesis) and from existing vessels
(angiogenesis) results in blood vessels lined by endothelial cells
(ECs).
[0086] Vascular endothelial growth factor (VEGF) is an interesting
inducer of angiogenesis and lymphangiogenesis because it is highly
specific endothelial cells. The VEGF family currently comprises
seven members: VEGF-A, VEGF-B, VEGF-C, VEGF-D, VEGF-E, VEGF-F, and
P1GF. All members have a common VEGF homology domain. Signal
transduction involves binding to tyrosine kinase receptors and
results in endothelial cell proliferation, migration, and new
vessel formation. In a preferred embodiment, VEGF (and/or other
known angiogenic or arteriogenic growth factors) is used to induce
EC differentiation, proliferation, infiltration, angiogenesis,
vascularization, etc., or any combination thereof.
[0087] It is not intended that the present invention be limited to
only one source or type of endothelial cell (EC). In one
embodiment, primary ECs are used in the open-top device. In one
embodiment, freshly isolated small vessel human dermal
microvascular endothelial cells are employed on the open-top-chip.
In one embodiment, an endothelial cell line is employed. In yet
another embodiment, human umbilical vein endothelial cells (HUVECs)
are used. In still another embodiment, bone marrow-derived
endothelial progenitor cells are seeded in the chip. In still
another embodiment, stem cells that can differentiate into ECs are
used.
[0088] It is not intended that the present invention be limited to
only one place for seeding the open-top-chip with ECs. While
placement of ECs on the underside of the membrane (in contact with
the fluidics) is preferred, placement on the topside of the
membrane and placement within the gel matrix itself are alternative
embodiments. With regard to the latter, in one embodiment,
microfluidic pathways in the gel itself are created that are
thereafter seeded with the endothelial cells.
[0089] For example, in one embodiment, microfluidic vessel networks
are engineered by seeding human endothelial cells [e.g. umbilical
vein endothelial cells (HUVECs)] into microfluidic circuits formed
via soft lithography in a type I collagen gel. Native, type I
collagen at 6-10 mg/mL is of an appropriate stiffness to allow high
reproducibility of vessel microstructure and also enables
remodeling through degradation and deposition of extracellular
matrix. The lithographic process enables the formation of
endothelium along the microfluidic channels and the incorporation
of living cells within the bulk collagen gel matrix within the
open-top-chip.
[0090] In one embodiment, endothelial cells are seeded into the gel
containing (or onto confluent lawns of) human fibroblasts and
cultured in the presence of high levels of ascorbate 2-phosphate to
create a tissue-like structure in which endothelial cells organize
into tube-like structures.
[0091] It is not intended that the skin model be limited to just
one type of keratinocyte. Indeed, the model can be used with many
types of cells of the integumentary system including but not
limited to Keratinizing epithelial cells, Epidermal keratinocyte
(differentiating epidermal cell), Epidermal basal cell (stem cell),
Keratinocyte of fingernails and toenails, Nail bed basal cell (stem
cell), Medullary hair shaft cell, Cortical hair shaft cell,
Cuticular hair shaft cell, Cuticular hair root sheath cell, Hair
root sheath cell of Huxley's layer, Hair root sheath cell of
Henle's layer, External hair root sheath cell, and Hair matrix
cells (stem cell). In one embodiment, human foreskin keratinocytes
are employed.
[0092] B. Other Cells and Tissues
[0093] A variety of different cells and tissue types can be modeled
and tested with the open-top spacer chip described herein. Indeed,
the system can virtually be adapted to all epithelial tissues. In
addition to skin, preferred models include (but are not limited) to
Lung, the Small Airway, the gut, muscle (including skeletal,
cardiac and or smooth muscle, and the Blood Brain Barrier (BBB).
Both human and animal cells are contemplated. Cell types which can
be used in the open-top devices include, but are not limited to Wet
stratified barrier epithelial cells, such as Surface epithelial
cell of stratified squamous epithelium of cornea, tongue, oral
cavity, esophagus, anal canal, distal urethra and vagina, basal
cell (stem cell) of epithelia of cornea, tongue, oral cavity,
esophagus, anal canal, distal urethra and vagina, Urinary
epithelium cell (lining urinary bladder and urinary ducts);
Exocrine secretory epithelial cells, such as Salivary gland mucous
cell (polysaccharide-rich secretion), Salivary gland serous cell
(glycoprotein enzyme-rich secretion), Von Ebner's gland cell in
tongue (washes taste buds), Mammary gland cell (milk secretion),
Lacrimal gland cell (tear secretion), Ceruminous gland cell in ear
(wax secretion), Eccrine sweat gland dark cell (glycoprotein
secretion), Eccrine sweat gland clear cell (small molecule
secretion), Apocrine sweat gland cell (odoriferous secretion,
sex-hormone sensitive), Gland of Moll cell in eyelid (specialized
sweat gland), Sebaceous gland cell (lipid-rich sebum secretion),
Bowman's gland cell in nose (washes olfactory epithelium),
Brunner's gland cell in duodenum (enzymes and alkaline mucus),
Seminal vesicle cell (secretes seminal fluid components, including
fructose for swimming sperm), Prostate gland cell (secretes seminal
fluid components), Bulbourethral gland cell (mucus secretion),
Bartholin's gland cell (vaginal lubricant secretion), Gland of
Littre cell (mucus secretion), Uterus endometrium cell
(carbohydrate secretion), Isolated goblet cell of respiratory and
digestive tracts (mucus secretion), Stomach lining mucous cell
(mucus secretion), Gastric gland zymogenic cell (pepsinogen
secretion), Gastric gland oxyntic cell (hydrochloric acid
secretion), Pancreatic acinar cell (bicarbonate and digestive
enzyme secretion), pancreatic endocrine cells, Paneth cell of small
intestine (lysozyme secretion), intestinal epithelial cells, Types
I and II pneumocytes of lung (surfactant secretion), and/or Clara
cell of lung.
[0094] One can also coat the membrane with Hormone secreting cells,
such as endocrine cells of the islet of Langerhands of the
pancreas, Anterior pituitary cells, Somatotropes, Lactotropes,
Thyrotropes, Gonadotropes, Corticotropes, Intermediate pituitary
cell, secreting melanocyte-stimulating hormone; and Magnocellular
neurosecretory cells secreting oxytocin or vasopressin; Gut and
respiratory tract cells secreting serotonin, endorphin,
somatostatin, gastrin, secretin, cholecystokinin, insulin,
glucagon, bombesin; Thyroid gland cells such as thyroid epithelial
cell, parafollicular cell, Parathyroid gland cells, Parathyroid
chief cell, Oxyphil cell, Adrenal gland cells, chromaffin cells
secreting steroid hormones (mineralcorticoids and gluco
corticoids), Leydig cell of testes secreting testosterone, Theca
interna cell of ovarian follicle secreting estrogen, Corpus luteum
cell of ruptured ovarian follicle secreting progesterone, Granulosa
lutein cells, Theca lutein cells, Juxtaglomerular cell (renin
secretion), Macula densa cell of kidney, Peripolar cell of kidney,
and/or Mesangial cell of kidney.
[0095] Additionally or alternatively, one can treat at least one
side of the membrane with Metabolism and storage cells such as
Hepatocyte (liver cell), White fat cell, Brown fat cell, Liver
lipocyte. One can also use Barrier function cells (Lung, Gut,
Exocrine Glands and Urogenital Tract) or Kidney cells such as
Kidney glomerulus parietal cell, Kidney glomerulus podocyte, Kidney
proximal tubule brush border cell, Loop of Henle thin segment cell,
Kidney distal tubule cell, and/or Kidney collecting duct cell.
[0096] Different geometries can be employed with dimensions related
to the different tissue types. For example, in one embodiment,
relatively tall spacer open top chip dimensions are contemplated
for the skin model, bronchial model, Kidney model and Gut model,
i.e. chamber height between 500 microns to 5 mm, chamber width 1
mm, chamber length 1.6 mm.
[0097] In another embodiment, relatively short spacer open top chip
dimensions are employed: chamber height between 100 to 500 microns,
chamber width 1 mm, chamber length 1.6 mm. These dimensions are
better suited to the Brain barrier and Lung models.
[0098] An example of the importance of its application is the small
airway model: Small Airway cells feel the paracrine stimulation of
neighbor cells, which stimulate their fully differentiation. In the
normal chip design cytokines are continuously flushed away from the
epithelial compartment by the constant flow, and this reduces or
impedes epithelial cell differentiation. The presence of this
porous matrix efficiently buffers the effect of flow reducing or
annul the effect of flow under cells.
[0099] The physical properties of the gels and fluids can vary (in
addition to the different geometries and dimensions for each of the
different tissue types). For example, for the Skin model and
bronchial model, a relatively high concentration collagen (8-11
mg/ml) is used. For the Kidney model and Gut model, a 1:1 mixture
of high concentration collagen:Matrigel is employed. For the Brain
barrier and lung models, a 1:1 low concentration (e.g. 3mg/ml) of
collagen/matrigel and/or fibronectin is employed. All in all,
concentrations above 0.3 mg/ml are required to form gels. Preferred
concentrations range between 3mg/ml and 10mg/ml. However,
concentrations above 5mg/ml are particularly suitable for use in
the open top chip.
[0100] Not all of the organ models require a gel. Indeed, some
organ chips are ideally used without a gel (e.g. lung). When gels
are used, more than one gel layer can be employed. For example,
hepatocytes can have a gel on both sides of the cells (e.g. a
matrigel layer on top and a collagen layer on the bottom.
Importantly, the gel can have a variety of thicknesses, including a
thin (molecular) coating. In one embodiment, the coating is made
with by ink jet printing.
[0101] Some cells do very well on patterned gels. For example,
muscle cells do well when they can deform to the surface. Indeed,
in one embodiment, the present invention contemplates a gel pattern
such that the sarcomeres align.
[0102] Importantly, the present invention contemplates
electrophysiological measurements in more than the blood brain
barrier (BBB) model. The present invention contemplates such
measurements for muscle (whether skeletal, cardiac or smooth
muscle) cells.
DETAILED DESCRIPTION OF THE INVENTION
[0103] While this invention is susceptible of embodiment in many
different forms, there is shown in the drawings and will herein be
described in detail preferred embodiments of the invention with the
understanding that the present disclosure is to be considered as an
exemplification of the principles of the invention and is not
intended to limit the broad embodiment of the invention to the
embodiment illustrated. For purposes of the present detailed
description, the singular includes the plural and vice versa
(unless specifically disclaimed); the word "or" shall be both
conjunctive and disjunctive; the word "all" means "any and all";
the word "any" means "any and all"; and the word "including" means
"including without limitation."
[0104] Those of ordinary skill in the art will realize that the
following description is illustrative only and is not intended to
be in any way limiting. Other embodiments will readily suggest
themselves to such skilled persons having the benefit of this
disclosure. Reference will now be made in detail to implementations
of the example embodiments as illustrated in the accompanying
drawings. The same or similar reference indicators will be used
throughout the drawings and the following description to refer to
the same or like items. It is understood that the phrase "an
embodiment" encompasses more than one embodiment and is thus not
limited to only one embodiment.
[0105] As used herein, the term "rigid" refers to a material that
is stiff and does not stretch easily, or maintains very close to
its original form after a force or pressure has been applied to it.
The term "elastomeric" as used herein refers to a material or a
composite material that is not rigid as defined herein. An
elastomeric material can be generally moldable, extrudable,
cuttable, machinable, castable, and/or curable, and can have an
elastic property that enables the material to deform (e.g.,
stretching, expanding, contracting, retracting, compressing,
twisting, and/or bending) when subjected to a mechanical force or
pressure and partially or completely resume its original form or
position in the absence of the mechanical force or pressure. In
some embodiments, the term "elastomeric" can also refer to a
material that is flexible/stretchable but it does not resume its
original form or position after pressure has been applied to it and
removed thereafter. The terms "elastomeric" and "flexible" are used
interchangeably herein.
[0106] The functionality of cells, tissue types, organs, or
organ-components can be implemented in one or more microfluidic
devices or "chips" that enable researchers to study these cells,
tissue types, organs, or organ-components outside of the body while
mimicking much of the stimuli and environment that the tissue is
exposed to in-vivo. In some embodiments, it is desirable to
implement these microfluidic devices into interconnected components
that can simulate the function of groups of organs,
organ-components, or tissue systems. In some cases it is desirable
to configure the microfluidic devices so that they can be easily
inserted and removed from an underlying fluidic system that
connects to these devices in order to vary the simulated in-vivo
conditions and organ systems (e.g., in situ conditions).
[0107] Many of the problems associated with earlier systems can be
solved by providing an open-top style microfluidic device that
allows topical access to one or more parts of the device or cells
that it comprises. For example, the microfluidic device can include
a removable cover, that when removed, provides access to the cells
of interest in the microfluidic device. In some embodiments, the
microfluidic devices include systems that constrain fluids, cells,
or biological components to desired area(s). The improved systems
provide for more versatile experimentation when using microfluidic
devices, including improved application of treatments being tested,
improved seeding of additional cells, and/or improved aerosol
delivery for select tissue types. In a preferred embodiment, the
open-top microfluidic device comprises a gel matrix.
[0108] The present disclosure additionally relates to
organ-on-chips ("OOCs"), such as fluidic devices comprising one or
more cells types for the simulation one or more of the function of
organs or organ-components. Accordingly, the present disclosure
additionally describes open-top organ-on-chips that solve problems
associated with earlier fluidic systems. Without limitation,
specific examples include models of skin, bronchial, and gut.
[0109] It is also desirable in some embodiments to provide access
to regions of a cell-culture device. For example, it can be
desirable to provide topical access to cells to (i) apply topical
treatments with liquid, gaseous, solid, semi-solid, or aerosolized
reagents, (ii) obtain samples and biopsies, or (iii) add additional
cells or biological/chemical components.
[0110] The present disclosure relates to fluidic systems that
include a fluidic device, such as a microfluidic device with an
opening that provides direct access to device regions or components
(e.g. access to the gel region, access to one or more cellular
components, etc.). Although the present disclosure provides an
embodiment wherein the opening is at the top of the device
(referred to herein with the term "open top"), the present
invention contemplates other embodiments where the opening is in
another position on the device. For example, in one embodiment, the
opening is on the bottom of the device. In another embodiment, the
opening is on one or more of the sides of the device. In another
embodiment, there is a combination of openings (e.g. top and sides,
top and bottom, bottom and side, etc.).
[0111] While detailed discussion of the "open top" embodiment is
provided herein, those of ordinary skill in the art will appreciate
that many embodiments of the "open top" embodiment apply similarly
to open bottom embodiments, as well as open side embodiments or
embodiments with openings in any other regions or directions, or
combinations thereof. Similarly, the device need not remain "open"
throughout its use; rather, as several embodiments described herein
illustrate, the device may further comprise a cover or seal, which
may be affixed reversibly or irreversibly. For example, removal of
a removable cover creates an opening, while placement of the cover
back on the device closes the device. The opening, and in
particular the opening at the top, provides a number of advantages,
for example, allowing (i) the creation of one or more gel layers
for simulating the application of topical treatments on the cells,
tissues, or organs, or (ii) the addition of chemical or biological
components such as the seeding of additional cell types for
simulating the function of tissue and organ systems. The present
disclosure further relates to improvement in fluidic system(s) that
improve the delivery of aerosols to simulate the function of tissue
and organ systems, such as simulated function of lung tissues.
[0112] Furthermore, the present disclosure contemplates
improvements to fluidic systems that include a fluidic device, such
as a microfluidic device with an open-top region that reduces the
impact of stress that can cause the delamination of tissue or
related component(s) (e.g., such as a gel layer).
[0113] Improvements to microfluidic devices for simulating the
function of a tissue are contemplated by the present disclosure
that include one or more of an open-top microfluidic device with
two or more chambers (e.g., microchannels) separated by a membrane.
In some embodiments, one or more of the devices further comprises a
gel in a chamber (e.g., microchannel or cavity) accessible through
an opening, including but not limited to an open-top structure, of
the microfluidic device. In some embodiments, the device further
comprises a removable or permanent cover for the microfluidic
device where the cover optionally has a fluidic chamber or
microchannel therein. Other desirable improvements that are
contemplated include a patterned gel in a microfluidic device.
[0114] The present disclosure further describes a method for
culturing cells in open-top devices. In some embodiments, the
method comprises placing a gel into an open-top structure. In some
embodiments, the method further comprises patterning the gel using
a shaping device, such as a patterned plunger stamp, a shaping
stamp, or similar devices. In some embodiments, the method
comprises permanently or reversibly applying a cover or other
shaping device to the open-top.
[0115] The present disclosure further relates to the use of fluidic
systems that include a fluidic device, such as a microfluidic
device with an open-top, to construct a model simulating the
structure and/or one or more functions of, for example, skin,
bronchial, or gut. In some embodiments, these models benefit from
the presence of gels, which for example, can provide a mechanical,
biochemical environment for one or more cells types, augment the
mass-transport characteristics, or provide an additional
compartment that may be used, for example, to house an additional
cell type (e.g. fibroblasts).
[0116] A system that provides for the use of a gel can be
particularly desirable for a skin model. For example, the current
state-of-the-art skin model, the living skin equivalent (LSE), is a
3D gel, 2 mm to 3 mm thick, that is embedded with fibroblasts with
differentiated keratinocytes on top of the gel. The actual
thickness of the gel can range from 0.1 mm to 5 mm. It is known
that a 3D gel is preferred to properly culture the fibroblasts
that, in turn, enables keratinocytes to fully differentiate. An
open-top architecture as described by some embodiments herein is
desirable because it enables LSE-like and similar cultures of
fibroblasts and keratinocytes, while further allowing the
introduction of an endothelial layer, the application of shear
forces, and the application of stretching to create a more
physiologically relevant model. Each of these optional features,
individually and collectively, provides desirable improvements over
current state-of-the-art LSE-like skin models.
[0117] Referring now to FIGS. 1 and 2, one type of a microfluidic
device referred to as an organ-on-chip ("OOC") device 100 is
illustrated that may be modified to include open-top embodiments
that are described in more detail later in this disclosure (see,
e.g., FIGS. 3-5 and 8-12). The OOC device 100 includes a body 112
that typically comprises an upper body segment 101 and a lower body
segment 103. The upper body segment 101 and the lower body segment
103 are typically made of a polymeric material, including, but not
limited to, PDMS (poly-dimethylsiloxane), polycarbonate,
polyethylene terephthalate, polystyrene, polypropylene,
cyclo-olefin polymers, polyurethanes, fluoropolymers, styrene
derivatives like styrene ethylene butylene styrene (SEBS), or other
polymer materials. The upper body segment 101, while illustrated
with a first fluid inlet 117 and a second fluid inlet 118, can be
modified to include an open region 104 (not shown) to optionally
allow the application of a gel layer 150 (not shown) to a membrane
140 and optionally modified to exclude the illustrated first fluid
inlet 117 and/or second fluid inlet 118. A first fluid path for a
first fluid includes the first fluid inlet 117, a first seeding
channel 127, an upper microchannel 134, an exit channel 131, and
then the first fluid outlet 124. A second fluid path for a second
fluid includes the second fluid inlet 118, a second seeding channel
128, a lower microchannel 136, an outlet channel 133, and then the
second fluid outlet 126.
[0118] Referring to FIG. 2, a membrane 240 extends between the
upper body segment 201 and the lower body segment 203. The membrane
240 is preferably an inert, polymeric, micro-molded membrane having
uniformly distributed pores with sizes normally in the range of
about 0.1 .mu.m to 20 .mu.m, though other pore sizes are also
contemplated. In some embodiments, the pore size is in the range of
about 0.1 .mu.m to 20 .mu.m. The overall dimensions of the membrane
240 include any size that is compatible with or otherwise based on
the dimensions of upper body segment 201 and lower body segment
103, such as about 0.05-100 mm (channel width) by about 0.5-300 mm
(channel length), though other overall dimensions are also
contemplated. In some embodiments, the overall dimensions of the
membrane 240 are about 1-100 mm (channel width) by about 1-100 mm
(channel length). In one embodiment, the thickness of the membrane
240 is generally in the range of about 5 .mu.m to about 500 .mu.m,
and in some embodiments, the thickness is about 20-50 .mu.m. In
some embodiments, the thickness can be less than 1 .mu.m or greater
than 500 .mu.m. It is contemplated that the membrane 240 can be
made of materials including, but not limited to
poly-dimethylsiloxane (PDMS), polycarbonate, polyethylene
terephthalate, styrene derivatives (e.g, styrene ethylene butylene
styrene, SEBS), fluoropolymers, and/or other elastomeric or rigid
materials. Additionally, the membrane 240 can be made of biological
materials including, but not limited to, polylactic acid, collagen,
gelatin, cellulose and its derivatives, poly(lactic-co-glycolic
acid), and/or comprise such materials in addition to one or more
polymeric materials. The membrane 240 separates an upper
microchannel 234 from the lower microchannel 236 in an active
region 237, which includes a bilayer of cells in the illustrated
embodiment. In some embodiments, a first cell layer 242 is adhered
to a first side of the membrane 240, and in some embodiments a
second cell layer 244 is adhered to a second side of the membrane
240. The first cell layer 242 may include the same type of cells as
the second cell layer 244. Or, the first cell layer 242 may include
a different type of cell than the second cell layer 244. And, while
a single layer of cells is shown for the first cell layer 242 and
the second cell layer 244, either the first cell layer 242, the
second cell layer 244, or both may include multiple cell layers or
cells in a non-layer structure. Further, while the illustrated
embodiment includes a bilayer of cells on the membrane 240, the
membrane 240 may include only cells disposed on one of its sides.
Furthermore, while the illustrated embodiment includes cells
adherent to the membrane, cells on one or both sides may instead be
not be adherent to the membrane as drawn; rather, cells may be
adherent on the opposing chamber surface or embedded in a
substrate. In some embodiments, the said substrate may be a
gel.
[0119] The OOC device 100 is configured to simulate a biological
function that typically includes cellular communication between the
first cell layer 242 and the second cell layer 244, as would be
experienced in-vivo within organs, tissues, cells, etc. Depending
on the application, the membrane 240 is designed to have a porosity
to permit the migration of cells, particulates, media, proteins,
and/or chemicals between an upper microchannel 234 and a lower
microchannel 36. The working fluids within microchannels 234and 236
may be the same fluid or different fluids. As one example, as OOC
device 100 simulating a lung may have air as a fluid in one channel
and a fluid simulating blood in the other channel. As another
example, when developing the cell layers 242 and 244 on the
membrane 240, the working fluids may be a tissue-culturing fluid.
Although it is not necessary to understand the mechanism of an
invention, it is believed that an organ-on-chip device offers
utility even in the absence of cells on one side of the membrane,
as the independent perfusion on either side of the membrane can
serve to better simulate the functions of mass-transport, shear
forces, and other embodiments of the biological environment. In one
embodiment, the active region 237 defined by an upper microchannel
234 and a lower microchannel 236 having lengths of about 0.1-10 cm,
and widths of about 10-2000 .mu.m.
[0120] The OOC device 100 preferably includes an optical window
that permits viewing of the fluids, media, particulates, etc. as
they move across the first cell layer 242 and the second cell layer
244. Various image-gathering techniques, such as spectroscopy and
microscopy, can be used to quantify and evaluate the effects of the
fluid flow in an upper microchannel 234 and a lower microchannel
236, as well as cellular behavior and cellular communication
through the membrane 240. More details on OOC devices can be found
in, for example, U.S. Pat. No. 8,647,861, and is incorporated by
reference in its entirety. Consistent with the disclosure in U.S.
Pat. No. 8,647,861, in one preferred embodiment, the membrane 240
is capable of stretching and expanding in one or more planes to
simulate functions of the physiological effects of expansion and
contraction forces that are commonly experienced by cells.
[0121] Micro- and mesofluidic devices and membranes can be
fabricated from or coated with or otherwise produced from a variety
of materials, including, but not limited to, plastics, glass,
silicones, biological materials (e.g., gelatin, collagen,
fibronectin, laminin, Matrigel.RTM., chitosan, and others).
[0122] Turning now to FIGS. 3 through 12 various exemplary open-top
microfluidic devices (e.g., open-top OOC devices) and components
are illustrated that can be used for creating gel layers, such as
for an open-top skin-on-a-chip device or for creating gel layers
for an open-top OOC device for simulating other biological
functions.
[0123] FIG. 3 illustrates an exploded perspective view of a
cross-section through an exemplary open-top microfluidic device 300
(e.g., an open-top OOC device). Open-top microfluidic devices, such
as an open-top OOC device, that allow access to the top of a chip
offer several benefits. Topical treatment, such as for a
skin-on-a-chip, can be applied directly through the open top to the
tissue of interest. Topical treatments can include, for example,
liquid, gas, gel, semi-solid, solid, particulate or aerosol.
Furthermore, additional chemical or biological components can be
added by means of the open top; as a particular example, additional
cell types can be seeded within the open top of the device. Aerosol
delivery, such as for a lung-tissue chip, is also contemplated and
can be completed through the open top, as well.
[0124] The microfluidic device 300 can optionally include a base
305, such as a glass slide, polymeric or metal support or a similar
structure, optionally providing an optical window. The base 305 can
support a bottom structure 325 of the microfluidic device 300. The
bottom structure 325 defines a bottom chamber 336 connected to a
bottom fluidic channel 409 in the microfluidic device 300. Above
the bottom structure 325 is a membrane 340 having a membrane top
side 348 and a membrane bottom side 349. The membrane bottom side
349 is disposed on the top surface of bottom structure 325 such
that membrane bottom side 349 rests above the bottom chamber 306. A
top structure 320 is disposed on the membrane top side 348 of
membrane 340 and defines an open region 304 for the open-top
microfluidic device 300 (e.g., the open-top chip). When the top
structure 320 is disposed on the membrane 340, it may be desirable
that all or substantially all of the open region 304 is bounded on
the bottom by the membrane top side 348 of the membrane 340.
[0125] In some embodiments, the chamber of the top structure 320
can further include a top microfluidic cover fluidic channel 308
(not shown) such as a top microfluidic cover fluidic channel 508
(e.g., as illustrated in FIG. 5A). In FIG. 5, such a top
microfluidic cover fluidic channel 508 may permit perfusion of a
top chamber 507, particularly while top chamber 507 is covered by
an optional fluidic cover 510 (FIG. 5B). In some embodiments, the
present invention contemplates that embodiments one or both of a
bottom structure fluidic channel 509 and a top microfluidic cover
fluidic channel 508 are microchannels. In some embodiments, the
present invention further contemplates that embodiments, an
optional fluidic covers, such as fluidic cover 410 or fluidic cover
510 (see, FIGS. 4 and 5, respectively) are disposed above a top
structure 520 and may further be in fluid communication with, and
define a top chamber 507 and an open region 504. Although it is not
necessary to understand the mechanism of an invention, it is
believed that a fluidic cover, such as fluidic cover 410, may be
designed for a one-time application (e.g. by means of bonding it in
place) or for subsequent removal.
[0126] An open region 304 in the open-top structure 320 may have
any shape, but is preferably a notch. In one embodiment, the
purpose of open region 304 is believed to allow direct access to
the membrane 340 or any matter disposed above it, before, during,
and/or after experimentation; such access is not available in
earlier closed microfluidic devices for simulating tissues. While
previous microfluidic devices, such as OOC, may have allowed for
low viscosity fluids to be directed through limited-access channels
to a membrane, such as illustrated in FIGS. 1 and 2, the open
region 304 in top structure 320 additionally allows for the
placement of high viscosity gels, high viscosity fluids, solids,
aerosols, and powders on an area of interest of membrane 340 (e.g.,
on the membrane inclusive of a predetermined tissue culture).
[0127] Turning now to FIG. 4, an exploded perspective view of an
exemplary open-top microfluidic device 400 includes a fluidic cover
410. The microfluidic device 400 includes an optional base 405 that
supports a bottom structure 425. The bottom structure 425 defines a
bottom chamber 406. Above the bottom structure 425 and the bottom
chamber 406 is an interface region 488 that comprises a membrane
440. The membrane 440 is disposed on the bottom structure 425 and
above the bottom chamber 406. A top structure 420 is disposed above
the membrane 440 and includes a top chamber 407 with an open region
404. When the top structure 420 is disposed on the membrane 440
during assembly of the device 400, it may be desirable that all or
substantially all of the open region 404 is bounded along the
bottom by the membrane 440.
[0128] The fluidic cover 410 may be designed to permit the
perfusion of the open region 404 while the fluidic cover 410 is
present. In some embodiments, the present invention contemplates
that this configuration provides an advantage over previous similar
devices that allows the perfusion of the open region 404 by way of
a top fluidic cover fluidic channel 408 in the top structure 420.
One of the benefits of including a top fluidic cover fluidic
channel 408 in the fluidic cover 410 instead of the top structure
420, is that cells, gel or other materials disposed in the open
region 404 are not allowed to leak or spread into the top fluidic
cover fluidic channel 408, where they may be undesirable. For
example, cells in the top fluidic cover fluidic channel 408 will
not be allowed to lie away from the active region 437 of membrane
440. In contrast, by disposing a top fluidic cover fluidic channel
408 in the fluidic cover 410, a benefit is provided of topfluidic
cover fluidic channels 408 being absent when a fluidic cover 410 is
removed, which disallows top fluidic cover fluidic channels 408
from being similarly filled with cells during seeding, as would
happen with channels being directly disposed in the top structure
420.
[0129] To minimize "leakage" of a substance of interest placed into
an open region 404 into areas where the substance is not desired,
different configurations of the open-top microfluidic device are
contemplated. For example, a fluidic cover 410 can include a top
chamber 407 (which may be a channel or part thereof) that
substantially aligns with all or a portion of the open region 404
cover disposed in top structure 420. The top chamber 407 may
optionally be hydraulically connected to one or more fluidic cover
inlet ports 414 and/or fluidic cover outlet ports 416 (see also,
fluidic cover inlet port 514, FIG. 5D), which in some embodiments
may be similar to the ports described for upper body segment 101 in
FIGS. 1 and 2. The presence of the top chamber 407 is especially
significant where the open region 404 is filled with a gel or other
substance that impedes fluid flow. In such a case, the top chamber
407 may be filled or perfused, enabling its contents to fluidically
interact with the substance in the open region 404. For example, if
the open region 404 holds a gel containing cells, flowing
tissue-culture media through the top chamber 407 (or even
incubating this media without flow) would allow nutrients and
reagents to be delivered to the cells, as well as for waste
products to be removed.
[0130] Through the use of a clamping device the fluidic cover 410
can be mechanically secured to the top structure 420 (e.g., see
FIG. 5E) to prevent or minimize leakage of any fluidic substance of
interest from the open region 404 of the open-top microfluidic
device 400. For example, a spring-loaded clamp can be used to
provide compression to a biocompatible polymer that uniformly seals
the open region without adhesives. Such sealing can be further
improved by including an elastomeric, pliable or soft material in
at least one of the fluidic cover 410 or top structure 420; one
with ordinary skill in the art will appreciate that many forms of
gasketing and sealing may be applied here. An advantage of some
embodiments that employ clamping is that they facilitate the
application, removal and potentially the reapplication of a lid or
cover, which may desirably allow access to the open region 404
after it was covered. Allowing access to an open region 404 of a
microfluidic device during experimentation can be useful, for
example, in (i) the application of topical treatment, aerosol,
additional cells or other biological reagents, (ii) change of
fluidic (e.g. tissue-culture media), (iii) sampling of fluidic or
solid matter, or (iv) imaging using optical or other techniques.
The option to reposition the cover or apply a different cover
further permits the continued use of the device (e.g. in a
biological experiment). Alternatively, the lid or cover may be
removed at the end of the device's use to permit sampling that may
be destructive, such as taking biopsies or otherwise removing
samples, staining, fixing, or imaging.
[0131] In some embodiments, the present invention contemplates that
a fluidic cover 410 can also, or alternatively, be bonded or
otherwise disposed onto the top structure 420. For example, for
fluidic or gas sealing, an adhesive membrane, laminate, film, or
sheet can be used to temporarily or permanently seal the open
region at the interface between the top structure that defines the
open region and a removable cover. It is also contemplated that
biocompatible polymer plugs or pistons can be used to seal off the
open region. It is further contemplated that an open region 404 of
an open-top microfluidic device 400 can be simply covered (e.g.,
similar to cell culture plates) with a cover or plate that limits
evaporation and improves sterile handling.
[0132] In embodimentsone embodiment, the present invention
contemplates that a top structure 420 can be used with an open
region 404, similar to a well, or with a removable fluidic cover
410 that may be akin to a flat layer that seals the top structure
420. An optional configuration in FIG. 4 includes a top chamber 407
with a fluidic cover fluidic channel 408 that can also introduce
fluids into the microfluidic device such as for perfusion or the
introduction of other liquids into the system.
[0133] As discussed above, open-top microfluidic devices described
herein offer a number of advantages. For example, these devices
allow the topical application of compounds to a membrane, including
compounds in the form or a gel or powder. The open-top design also
allows for aerosol delivery to effect a simulated function of a
tissue directly from the top of the microfluidic device.
Furthermore, the open-top configuration allows access to apply
simulated effects of wounding to a tissue (e.g., simulate effects
of a burn or scratch on the skin or intestine) during the course of
testing and the application of a treatment of interest all within
the same microfluidic device and as part of the same
experimentation cycle.
[0134] Furthermore, the open-top configurations described herein
also allow direct access to the epithelium, and thus, allow the
ability to biopsy a sample during testing. An open-top
configuration also allows microscopy to be applied during use of a
chip, such as the application of electron microscopy,
high-magnification imaging methods, and laser-based imaging methods
by removing the top cover of the microfluidic device, while
optionally maintaining the integrity of the experiment.
[0135] In some embodiments, it is desirable to simulate one or more
functions of lung, as such function simulations may be beneficial,
for example, in testing compound transport and absorption through
the lung, the effect of aerosolized or inhaled compounds, model
lung disease, or otherwise observe lung response. In vitro models
are known in the art, including for example a lung-on-a-chip
microdevice disclosures in U.S. Pat. No. 8,647,861, entitled,
"Organ Mimic Device with Microchannels and Methods of Use and
Manufacturing Thereof," and the small-airway on-a-chip microdevice
disclosures in International Publication No. WO 2015/0138034,
entitled, "Low Shear Microfluidic Devices and Methods of Use and
Manufacturing Thereof," both of which are hereby incorporated by
reference herein in their entireties. A lung model that combines
several of desired features in the same model would be beneficial.
Desired features include recapitulation of various elements of lung
structure and morphology, and the ability to satisfactorily
introduce compounds or materials as aerosols, fluidic access (e.g.
to emulate blood or air flow), or mechanical forces. For example, a
lung model is desirable that minimizes loss of aerosol that can
occur in delivery tubing and channels and variation in the aerosol
delivery along the length of the channel. According to some
embodiments of the present disclosure, a lung model that includes
one or more of such desired features can be constructed. For
example, in one embodiment, a lung module is constructed using an
open-top device, such as that illustrated in FIG. 4 (whether
employing a fluidic cover 410, the optional cover of FIG. 3, or no
cover). Accordingly, lung epithelial cells (e.g. alveolar
epithelial cells) can be included or deposited within the open
region 404. Optionally, the bottom structure 425 may include
endothelial cells, motivated by the presence of similar cells in
the vasculature (e.g. capillary bed) of an in vivo lung. It is also
contemplated that using the various embodiments of open-top devices
described herein, a lung model may be biologically cultured or
operated statically (i.e., for example, without continuous flow or
with discrete exchanges of some portion of the liquid in the
device) or under flow in either fluidic channels disposed in, for
example, the bottom structure 425, top structure 420, or cover 410,
as well as any combination of these modalities, which may
optionally be varied during operation (e.g. begin with discrete
fluid exchanges, then introduce flow). In addition, the open region
404 or cell layers within it may be cultured dry, under an
air-liquid interface, or submerged, with this mode of culture
optionally varied during use. For example, following the example of
the lung-on-a-chip and small-airway-on-a-chip devices, it may be
desirable to begin lung culture under submerged conditions and
transition to an air-liquid interface culture after some maturation
period (e.g. ranging without limitation from 1 hour to 7 days, or
from 1 day to 14 days).
[0136] A particular advantage of the various open-top embodiments
of the present disclosure is that aerosol may be delivered to the
lung cells in the open region, such as open region 404. In one
exemplary embodiment, while operating the device without the
optional cover (or by removing the cover), aerosol can be delivered
directly into the open region 404 from above (or substantially
above). The aerosol may be generated using any of a variety of
aerosol-generation techniques known in the art. Alternatively, an
aerosol generation means may be included in a cover that can be
placed on top of the open region 404. A cover may be optionally
removed or exchanged during use; for example, an aerosol-generating
cover may be applied when aerosol is desired and replaced with a
fluidic cover 410 when fluidic perfusion is desired. In some
embodiments, non-aerosol materials or samples can be applied to
cells present in an open region, such as open region 404. This may
include, but are not limited to, materials or samples that are
difficult to apply fluidically due to their properties, such as
slurries, pastes, solids, or viscous fluids.
[0137] Referring now to FIGS. 5A-5F, multiple perspective views,
including additional cross-sectional details through an exemplary
open-top microfluidic device, are illustrated. The microfluidic
device 500 includes a membrane 540 disposed between a bottom
structure 525 and a top structure 520. The bottom structure defines
a bottom chamber 506, ' and the top structure 520 includes a top
chamber 506 that defines an open region 504, of the microfluidic
device 500. In some embodiments, it is desirable that the open
region 504, includes a gel layer 550, comprising a porous volume,
or another material for testing (e.g., an extracellular matrix or
cells embedded in an extracellular matrix). For example, a gel
layer 550 can include gels used in an organ-on-chip model of the
skin to house fibroblasts and to support a layer or keratinocytes.
In FIG. 5B, a gel layer 550 is introduced into the open region 504
(see FIG. 5A) where the gel layer 550 is bounded on the bottom by
membrane 540.
[0138] In some embodiments, a gel layer 550, or porous volume, is
formed by injecting one or more suitable precursors through one or
more fluidic channels included in the top structure 520 (such
optional channels are depicted in FIGS. 5A-5C). The one or more
precursors can then be treated as desired to form the gel or porous
volume (e.g. UV light, chemical treatment, temperature treatment
and/or incubation/waiting). Alternatively, the one or more
precursors are in a final or near-final form, where no additional
active process is applied in order to generate the gel or porous
volume. While the approach of injecting the one or more precursors
through one or more fluidic channels included in the top structure
520 can be adapted to permit consistent filling with gel or other
porous volume, it typically results in the gel or porous volume
filling at least part of the said fluidic channels. This may be
undesirable in some situations; for example, when dealing with a
gel containing cells, it is desirable to limit the cells to the
active region, lest they may not receive sufficient nutrient or
biochemical cues through the membrane.
[0139] Alternatively, the one of more precursors can be placed into
the top of the open-top microfluidic device via the open region
504. Such an approach permits alternative embodiments that
eliminate or limit spaces into which the precursors may spread
(e.g. one may avoid fluidic channels included in the top structure
520 that are in fluidic communication with the open region 504). In
other embodiments, the one or more precursors may be injected into
the open region 504, by means of a fluidic cover 510 that includes
one or more fludic cover fluidic channels 508 (an example is
illustrated in FIG. 5D). Although such embodiments may also result
in a gel layer 550 formed in the fluidic cover fluidic channels
508, the fluidic cover 510 can be removed and optionally replaced,
removing at least part of the undesired material.
[0140] In some embodiments, it is desirable to limit or shape the
gel volume or porous volume. For example, in an organ-on-chip model
of the skin, it is may be desirable to limit the thickness of a gel
layer housing fibroblasts and supporting keratinocytes to a
selected thickness. Without limitation, such thickness may be
chosen from one or more of the ranges of 10 .mu.m to 200 .mu.m, 100
.mu.m to 1 mm, 0.5 mm to 5 mm, or 1 mm to 10 mm. According to some
embodiments, the extent of a gel layer 550, or porous volume, may
be limited by a shaping device 559 (e.g., a shaping cover, a
plunger 560 with a patterned base) that is present during the
introduction or formation of the gel or porous volume. This shaping
device 559 may be removed and optionally replaced with a cover
(e.g., a fluidic cover 510) once a gel layer 550, or porous volume,
has formed. The shaping device 559 may optionally include a chamber
into which the gel or porous volume can conform, at least in part.
Alternatively, a shaping device 559 may include one or more
features that protrude into the open region 504. FIG. 5C
illustrates one type of a shaping device with features that
protrude into the open region 504, which takes the form of a
plunger stamp 560. In some embodiments, shaping devices are applied
before the introduction of one or more precursors for a gel or
porous volume; for example, it could be introduced through fluidic
channels present in the top structure 520, a fluidic cover 510 or
even in the shaping device itself. In other embodiments, the one or
more precursors are introduced before the application of the
shaping device, whether through fluidic channels in the top
structure 520 or fluidic cover 510, or introduced directly into the
open region 504 (e.g. using a syringe, pipette or printing
process). In such cases, the shaping device may optionally include
features (e.g. holes, fluidic channels, cavities) designed to allow
the capture of excess precursor. In some embodiments, the shaping
device comprises a plurality of layers. For example, the shaping
device may include a spacer layer used to define gel height and a
flat cover to prevent the gel from passing the spacer's height. All
or only a subset of these layers may be removed once the gel or
porous volume is defined, with the remaining layers (e.g. spacer
layer) potentially remaining during device use or experimentation.
In some embodiments, the top structure 520 may be removed after gel
or porous volume formation, and can be optionally replaced with a
different structure or cover, that may or may not include an open
region.
[0141] In one embodiment, the present invention contemplates a gel
layer 2050 comprising a plurality of gel micropillars 2053. FIG.
20A. For example, such gel micropillars 2053 may be arranged in
symmetrical rows along the surface of the gel layer 2050. FIG.
20B.
[0142] In one embodiment, the present invention contemplates a gel
layer formed as a gel mesh 2054. FIG. 20A. For example, such a gel
mesh 2054 may be formed as an insert within a top chamber 2006 or
bottom chamber 2007. FIG. 20B.
[0143] In one embodiment, the present invention contemplates a
shaping device comprising a plunger stamp 560having a patterned
surface 665 that creates a pattern in the gel or porous volume at a
patterning interface 555. Depending on the properties of the
precursor materials (e.g. viscosity of the precursor and its change
through curing), the shaping device may be removed before the gel
or porous volume have fully formed.
[0144] FIG. 5D next illustrates a perspective view of the exemplary
open-top microfluidic device of FIG. 5C after a plunger stamp 560
has been removed, including a patterned top surface 557 in the gel
layer 550. The patterning includes depressions 558 in the patterned
top surface 557 of the gel layer 550. The removable fluidic cover
510 can then be placed onto microfluidic device 500 such that top
chamber 507 aligns with bottom chamber 506. An exemplary fluidic
cover 510 can optionally include fluidic channels. In the example
illustrated, one of the fluidic channels 508 extends from inlet
hole 514 to the top chamber 507. An outlet fluidic chamber 515 ends
at outlet hole 516 wherein the outlet fluidic chamber 515 extends
downwardly through the fluidic cover 510, and connects through an
opening in the membrane 540, such that it is fluidically connected
with chamber 506. The fluidic cover 510 may be removable, and once
removed it may be optionally reapplied or optionally replaced with
a different cover.
[0145] FIG. 5E illustrates the exemplary open-top microfluidic
device disposed within an exemplary clamping device 570. A clamping
device 570 can be desirable because no glue or bonding is needed to
hold the various layers of the microfluidic device together. The
clamping device applied to an open-top microfluidic device
optionally allows efficient removal of the removable cover during
an experiment. The clamping device 570 for the microfluidic device
500 can include an optional platform 585 for engaging a first side
(e.g., the bottom side) of the microfluidic device 500. In some
embodiments, a plurality of elongated posts 590 can extend upwardly
from the platform 585. A compression plate 580, which may flat or
may in some embodiments be uneven, is movably coupled to the
plurality of elongated posts 590 such that the compression plate
580 is vertically slidable along the posts 590. In some
embodiments, the compression plate 580 engages a second side (e.g.,
the top side) of the microfluidic device 500; in other embodiments,
the compression plate 580 retains a cover to the microfluidic
device 500. A compression device 580 provides compressive forces
(e.g., see arrows 598) generally in a direction along the elongated
posts 590. The compression device (e.g., springs 595, elastomers,
flextures, etc.) is operatively connected to the compression plate
580 such that the compressive forces (e.g., see arrows 598) create
a substantially uniform pressure on the second side (e.g., the top
side) of the microfluidic device 500. Clamping device components
can be made from different types of materials, including, but not
limited to, PMMA (e.g., acrylic), thermoplastics, thermoset
polymers, other polymer materials, metals, wood, glass, or
ceramics. In alternate embodiments, the compressive plate 580 may
be held in place using a retention mechanism including, but not
limited to, one or more of screws, clips, tacky/sticky materials,
other retention mechanisms known in the art, or the combination of
any of these mechanisms and/or the aforementioned compression
device. In some embodiments, a retention mechanism retains a
compressive plate 580 with respect to or against a platform 585. In
alternate embodiments, a retention mechanism retains a compression
plate 580 with respect to or against a microfluidic device 500. For
example, screws can be used to fasten a compression plate 580
against a microfluidic device 500 with a corresponding threaded
holes included in a microfluidic device 500. As another example, a
compression plate 580 can include a clip feature (as a retention
mechanism) that clips into a suitable receiving feature of a
microfluidic device. In some embodiments, thae compression plate
580 comprises a cover for an open area included in a microfluidic
device 500. In other embodiments, a compression plate 580 retains
an additional substrate that comprises a cover for an open area
included in a microfluidic device 500.
[0146] In some embodiments, a compression plate 580 may include at
least one access hole 581 that substantially aligns with a
corresponding fluid port (e.g., inlet hole 514 or outlet hole 516)
on a microfluidic device 500 or an optional cover. In some
embodiments, an access hole 581 securely holds or comprises a fluid
connector. Such a fluidic connector may be beneficial in
fluidically interfacing with a microfluidic device 500 or optional
cover without necessitating that a connector be included in a
microfluidic device 500 or optional cover.
[0147] A bottom surface area of the compression plate 580 may be
greater or smaller than a top surface area of the microfluidic
device 500. In some embodiments, the platform 585 can have a width
such that the compression plate width is greater than the base
width. The compression plate 580 can further include finger nubs or
tabs (not shown) protruding from a central portion of the
compression plate and extending beyond the base such that a
compression plate width with the finger nubs is greater than the
base width.
[0148] In embodiments that include elongated posts 590, it is
contemplated that the plurality of elongated posts 590 are
substantially parallel and the compression plate 580 includes a
plurality of apertures operative to allow an elongated post to pass
through a respective aperture. The plurality of elongated posts 590
supports the compression device (e.g., springs 595). The
compression device can include at least one spring 595 extending
around an outer boundary of at least one of the plurality of
elongated posts 590. In some embodiments, a compression plate 580
comprises two springs 595 that provide a substantial uniform or
equalized pressure to a compression plate where a compression plate
is a mobile part of the clamping device 570 that moves easily up
and down (or along other axes) to allow for easy manipulation of
the clamped system. For example, the use of springs in a clamping
device can be desirable because springs constants can provide for a
wide range of translation distances and forces and are versatile
for situations where a clamping device may be positioned upside
down for extended periods of time. A compression plate 580 can be
modified in area, shape, thickness, or material.
[0149] Although it is not necessary to understand the mechanism of
an invention, it is believed that a maximum compressive force
provided to a microfluidic device by a clamping device is
determined based on the force required to create a fluidic seal
between a compression plate 580 or optional cover and a
microfluidic device 500 (if such a seal is desired), and a
propensity for the collapse of microfluidic channels or chambers
within the microfluidic device 500 or optional cover. In some
embodiments, compressive forces provided can range from
approximately 50 Pa (approximately 0.007 psi) to approximately 400
kPa (approximately 58 psi). In some embodiments, compressive forces
provided can range from approximately 5 kPa (0.7 psi) to
approximately 200 kPa (29 psi). In some embodiments, it is
desirable that the amount of force or pressure applied by a
compression plate 580 to a microfluidic device 500 keep a
microfluidic device sealed or properly sandwiched between the
compression plate 580 and a platform 585 while not being so extreme
as to cause the collapse of the microfluidic channels or to prevent
desired gas exchange.
[0150] A glass slide or other transparent window (e.g. made of
PMMA, polycarbonate, sapphire, etc.) can be integrated into a
clamping device 570 to provide a rigid support for the microfluidic
device which improves pressure distribution for flexible devices
(such as those made from PDMS silicone) while enabling good optical
access for macroscopic, visual, or microscopic imaging that may be
desirable through viewing portions of the clamp system.
[0151] In one embodiment, the present invention contemplates that
the described clamping device can facilitate the use or positioning
of the device in an upside down position. This can be a
particularly desirable feature during cell seeding of the underside
of a chip membrane, commonly done during OOC co-culture. A
compression device for the clamping device 570 can include
alternatives to springs or other aforementioned compression devices
or retention mechanisms. For example, hydraulic or pneumatic
compression systems are contemplated. It is also contemplated that
for rigid microfluidic devices compliant gaskets can be used. For
example, the clamping device 570 can be fitted with a compliant
gasket that has a level of springiness to it rather than a spring
itself. The compliant gasket materials would create an interface
between the compression plate 580 and the microfluidic device 500
or between an optional cover and the microfluidic device 500. It is
also contemplated that in some embodiments a compression device can
utilize geometric shapes, such as cantilevered beams, as part of
the device design to provide compressive force resulting from the
case material flexure or compression. In some embodiments, the
compressive force can also be provided with magnetic or
electromagnetic systems.
[0152] FIG. 5F illustrates a perspective view of an alternative
exemplary cross-section through an open-top microfluidic device,
similar to device 500, with a bottom chamber 506 and open region
504 that are generally circular from a top or bottom view
perspective. Other embodiments can include an oval or football
shape. Another exemplary feature includes a membrane 540 disposed
between the bottom structure 525 and the top structure 520, where
the bottom structure defines the bottom chamber 506 and the top
structure defines the open region 504. The illustrated membrane 540
limits passage between the channels (e.g., the open region 504 and
the bottom chamber 506) to a plurality of holes 541 that in some
embodiments comprise less than the entire surface area of the
membrane 540 within the open region 504 and bottom chamber 506. The
plurality of holes 541 may include laser cut holes for passage of a
gel, a porous volume, or another material (e.g., an extracellular
matrix or cells embedded in an extracellular matrix) that has been
disposed in the open region for testing.
[0153] In some embodiments, an open-top microfluidic device allows
for the direct deposition of a matrix, for example a gel or a
porous volume or a biodegradable polyester such as polycapolactone,
into the open region or open portion of an open-top microfluidic
device. For example, a gel-forming solution or precursor can be
placed in a mold that is separate from the microfluidic device. The
mold can approximate the shape of the chamber or open region into
which the gel volume will be disposed for a desired experiment.
Similar to setting a gel layer 550 directly into the microfluidic
device 500 (see FIGS. 5C-5D), a plunger stamp 560 is placed into
the gel solution in the mold such that a bottom surface of the
plunger stamp is in contact with the gel solution in the mold. The
bottom surface of the plunger stamp includes the pattern of
features 555 for imprinting into the gel solution. After the gel
solution has at least partially solidified, the plunger stamp is
then removed from the gel solution, thereby creating a patterned
gel surface 557 to simulate the functions of a tissue
microstructure. Once the gel has solidified to the point where the
gel will not break apart or otherwise separate, the patterned gel
can be removed from the mold and be inserted into the similarly
shaped open region of the actual microfluidic device to be used for
experimentation. Alternatively, or in combination, a suitably
shaped volume or gel or porous volume can be cut to size, 3D
printed or aggregated from smaller volumes, then disposed into the
open region. Further, a gel or porous volume can be 3D printed
directly into the open region. In another related embodiment, a
matrix (e.g., gel or porous volume) such as one formed as described
for FIGS. 5C-5D, can also be easily extracted (whether whole or in
part) from the top structure of an open-top microfluidic device,
which provides benefits by overcoming the problem of staining and
high-resolution imaging without having to stain an entire chip or
having to reconstruct cell-monolayers. The removal or insertion of
a gel, porous material and/or biological sample (e.g. biopsy,
blood) to or from the open region of an open-top microfluidic
device is also desirable because it can allow access for testing of
the subject tissue sample in the microfluidic device and/or then
the subsequent removal of the sample from an OOC device, which can
then be used for other applications (e.g., for implantation into a
patient; additional analysis in another device). In an alternative
embodiment, the gel or gel containing cells or tissue can be
patterned following culture of cells in the gel material.
[0154] In some embodiments of a microfluidic device, it is
desirable to include a cover that comprises sensors or actuators.
For example, a cover can comprise one or more electrodes that can
be used for measurement of electrical excitation. In some
embodiments, such as where the device comprises a membrane (e.g.,
membrane 540), the one or more electrodes can be used to perform a
measurement of trans-epithelial electrical resistance (TEER) for
the membrane. It may also be desirable to include one or more
electrodes on the opposite side of the membrane 540. In some
embodiments, the electrodes can be included in a bottom structure
(e.g., bottom structure 525). In some embodiments, the bottom
structure can be an open bottom with bottom electrodes included on
a bottom cover that can be brought into contact with the bottom
structure. The bottom cover may support any of the features or
variations discussed herein in the context of a top cover,
including, for example, removability, fluidic channels, multiple
layers, clamping features, etc.
[0155] In some embodiments, it is desirable to simulate one or more
functions of skin, for example, in testing compound transport and
absorption through the skin, the effect of topical treatments on
skin aging or healing, modeling skin disease, or observing skin
response such as damage or sensitization. While in vitro skin
models are known, such as living skin equivalent (LSE), a skin
model that combines several features in the same model would is
desirable. For example, desirable features can include
recapitulation of various elements of skin structure and
morphology, topical access, fluidic access (e.g. to emulate blood
flow), or mechanical forces. According to some embodiments of the
present invention, a skin model that includes one or more of such
desired features can be constructed. In one exemplary embodiment,
the skin model is constructed using the open-top device illustrated
in FIG. 5D. Accordingly, a gel layer 550, which may be considered
to correspond to the skin's dermal layer, is present in or
introduced into (e.g. using any of the aforementioned methods) the
open region 504. Optionally, the gel layer 550 (or other matrix)
may include embedded fibroblasts or related cells, motivated by the
presence of similar cells in the dermal layer of in vivo skin.
Furthermore, the gel layer 550 is topped by keratinocytes, which
are a primary cell type of the skin. The keratinocytes may, for
example, be deposited on top of the gel layer 550 (which can be
done, for example, directly through the open top or introduced
fluidically through channels present in the top structure 520 or
cover 510) or present in the gel or other device component and
allowed to biologically mature or develop into a cell layer at the
top of the gel layer 550. Optionally, the bottom structure 525
includes endothelial cells, motivated by the presence of similar
cells in the vasculature (e.g. capillary bed) of in vivo skin.
Using various embodiments of the open-top device described herein,
the resulting skin model may be biologically cultured or operated
statically (i.e., for example, without continuous flow or with
discrete exchanges of some portion of the liquid in the device) or
under flow in either fluidic channels disposed in the bottom
structure 525 top structure 520 or cover 510, as well as any
combination of these modalities, which may optionally be varied
during operation (e.g. begin with discrete fluid exchanges, then
introduce flow). In addition, the open region 504 or cell layers
within the open-top microfluidic device may be cultured dry, under
an air-liquid interface, or submerged, with this mode of culture
optionally varied during use. For example, following the example of
prior skin models such as the LSE, it may be desirable to begin
keratinocyte culture under submerged conditions and transition to
an air-liquid interface culture after some maturation period (e.g.
ranging without limitation from 1 hour to 3 days, or from 1 day to
14 days). The gel layer 550 may comprise a biological or synthetic
gel or other porous volume, including for example, collagen I,
collagen IV, fibronectin, elastin, laminin, gelatin,
polyacrylamide, alginate, or Matrigel.RTM.. Collagen I in
particular has been used by prior skin models, whereas it is known
that elastin is present in in vivo skin, motivating its use in the
disclosed in vitro model.
[0156] In some embodiments, it can be similarly desirable to
simulate one or more functions of the intestine, for example, in
testing compound transport and absorption through the intestine or
its parts, the effect of treatments on intestine health or healing,
modeling intestinal disease, or observing intestinal response such
as damage or sensitization. In vitro intestinal models are known in
the art, including for example transwell-based systems or the
gut-on-a-chip microdevice disclosures in U.S. Patent Publication
No. 2014/0038279, entitled "Cell Culture System, " which is
incorporated by reference herein in its entirety. In some
embodiments, it is desirable construct an intestinal model that
combines several of the desired features in the same model,
including recapitulation of various elements of intestinal
structure and morphology, fluidic access (e.g. to emulate luminal
transport or blood flow), or mechanical forces. According to some
embodiments of the present disclosure, an intestine model that
includes one or more of such desired features can be constructed.
In one exemplary embodiment, the intestine model is constructed
using the open-top device illustrated in FIG. 5D. Accordingly, a
gel layer 550, is present in or introduced into (e.g. using any of
the aforementioned methods) the open region 504. Furthermore, the
gel layer 550 is topped by intestinal epithelial cells. The
intestinal epithelial cells may, for example, be deposited on top
of the gel layer 550 (which can be done, for example, directly
through the open top or introduced fluidically through channels
present in the top structure 520 or cover 510) or be present in the
gel or other device component and allowed to biologically mature or
develop into a cell layer at the top of the gel layer 550.
Optionally, the bottom structure 525 includes endothelial cells,
motivated by the presence of similar cells in the vasculature (e.g.
capillary bed) of in vivo intestines. Optionally, the gel layer 550
includes cells, for example, smooth muscle cells, neuronal cells,
lymphatic cells or other cells types, cultures within the gel layer
550. Using various embodiments of the open-top device described
herein, the resulting model may be biologically cultured or
operated statically (i.e., for example, without continuous flow or
with discrete exchanges of some portion of the liquid in the
device) or under flow in either fluidic channels disposed in the
bottom structure 525 top structure 520, or cover 510, as well as
any combination of these modalities, which may optionally be varied
during operation (e.g. begin with discrete fluid exchanges, then
introduce flow). Although cells of the intestine are typically
cultured submerged, the open-top device also permits the open
region 504 or cell layers within it to be cultured dry or under an
air-liquid interface, to simulate intestinal gas or various
pathologies (e.g. swallowed air or gas presence with irritable
bowel syndrome or lactose intolerance), or cultured with highly
viscous or solid particulate material (e.g., food, fecal matter,
etc.) with the mode of culture optionally varied during use. The
gel layer 550 may comprise a biological or synthetic gel or porous
volume, including for example, collagen I, collagen IV,
fibronectin, elastin, laminin, gelatin, polyacrylamide, alginate,
or Matrigel.RTM..
[0157] It some embodiments, it can be similarly desirable to
simulate one or more functions of the small airway, for example, in
testing compound transport and absorption through the airway or its
parts, the effect of treatments on airway health or healing,
modeling airway disease, or observing airway response such as
damage or sensitization. In vitro small airway models are known in
the art, including for example the small-airway on-a-chip
microdevice disclosures in International Publication No. WO
2015/0138034, entitled, "Low Shear Microfluidic Devices and Methods
of Use and Manufacturing Thereof," which is hereby incorporated by
reference herein in its entirety. According to some embodiments of
the present disclosure, a small-airway model can be constructed to
include one or more desired features, including for example fluidic
access to airway and vasculature, several of the differentiated
cell types found in the in vivo airway (e.g. ciliated cells,
mucus-producing cells), and immune response. In one exemplary
embodiment, the small-airway model is constructed using the
open-top device illustrated in FIG. 5D.
[0158] Accordingly, a gel layer 550, is present in or introduced
into (e.g. using any of the aforementioned methods) the open region
504. Furthermore, the gel layer 550 is topped by small-airway
epithelial cells. The small-airway epithelial cells may, for
example, be deposited on top of the gel layer 550 (which can be
done, for example, directly through the open top or introduced
fluidically through channels present in the top structure 520 or
cover 510). Optionally, the bottom structure 525 includes
endothelial cells, motivated by the presence of similar cells in
the vasculature (e.g. capillary bed) of in vivo airway. Using
various embodiments of the open-top device described herein, the
resulting model may be biologically cultured or operated statically
(without continuous flow or with discrete exchanges of some portion
of the liquid in the device) or under flow in either fluidic
channels disposed in the bottom structure 525, top structure 520,
or cover 510, as well as any combination of these modalities, which
may optionally be varied during operation (e.g. begin with discrete
fluid exchanges, then introduce flow). In addition, the open region
504 or cell layers within it may be cultured dry, under an
air-liquid interface, or submerged, with this mode of culture
optionally varied during use. The gel layer 550 may comprise a
biological or synthetic gel or porous volume, including for
example, collagen I, collagen IV, fibronectin, elastin, laminin,
gelatin, polyacrylamide, alginate, or Matrigel.RTM..
[0159] In some embodiments, it is desirable to provide mechanical
strain or force to at least a portion of the fluidic device. In
particular, it may be desirable to apply mechanical force to at
least some cells present within the fluidic device. According to
some embodiments, a mechanical force is applied to at least one
portion of an open-top device by incorporating an actuation
mechanism. In some embodiments, this actuation mechanism can
include one or more operational channels, similar to ones described
by U.S. Pat. No. 8,647,861, which is hereby incorporated by
reference herein in its entirety. Such operational channels can be
evacuated or pressurized to cause the application of force to a
portion of the device, for example, a membrane separating a top and
bottom fluidic channels. In this example, any cells present on top
or below the membrane may experience the mechanical force, leading
to a potential biological effect. In some embodiments, an open-top
device is included in a system that additionally includes an
actuation mechanism. In some embodiments, this actuation mechanism
comprises a system for mechanically engaging the open-top device
and a system for applying a stretch or compression force. A number
of examples of actuation systems included in a fluidic device or in
systems that include a fluidic device are described by
International Application No. PCT/US2014/071570, filed Dec. 19,
2014, entitled "Organomimetic Devices and Methods of Use and
Manufacturing Thereof", which is hereby incorporated by reference
herein in its entirety. In one exemplary embodiment, a system
comprises an open-top device, a mechanical engaging device
including one or more clamps or pins, and a mechanical actuation
device including one or more electrical motors or pneumatic
cylinders. According to one method to employ such a system, the
open-top device is engaged with the mechanical engaging mechanism
(e.g. by slipping the one or more pins into corresponding holes
included in the open-top device), and actuating said one or more
electrical motors or pneumatic cylinders to apply a cyclical
mechanical force on at least part of the open-top device.
[0160] Turning now to FIG. 6, another exemplary shaping device 560
(in this case a plunger stamp 660) with a textured bottom surface
666 is illustrated for simulating biological conditions in an
open-top microfluidic device (e.g., an open-top OOC device). The
plunger stamp 660 can be used in a similar manner as illustrated in
FIG. 5C and 5D. Plunger stamps can also be used to create gel
layers of a defined thickness in the open region of an open-top
microfluidic device. This can be particularly beneficial where a
separate section or layer may be needed to introduce a dermal
equivalent layer, such as a collagen plus a fibroblast. A plunger
stamp can also be beneficial for skin development in, for example,
an open-top OOC device, by allowing the creation of a thick gel
layer (e.g., about 50 micrometers to about 10 millimeter thick,
about 100 micrometers to about 1 millimeter thick), such as for an
in vivo skin section. The plunger stamp can also be used in
applications where cells are embedded into a system, such as an ECM
with the introduction of cells into the matrix. Application of a
plunger stamp to a gel in an open region of an open-top
microfluidic device also allows for the embedding of fibroblasts
into the gel layer.
[0161] Patterned surfaces created with a shaping device (e.g.
plunger stamp) can provide for more accurate simulation of tissue
or organ characteristics, such as for skin tissue, small-airway
tissue and intestine. For example, a gel layer for a skin model can
be formed to be undulating, with the undulations mimicking features
of in vivo papillae or rete peg structures. Such structures are
hallmarks of in vivo skin and can vary with skin health and age.
Accordingly, the ability to form and control structures in the
open-top chip that mimic in vivo structures is a beneficial
embodiment of the disclosed open-top microfluidic systems. As a
further example, patterning using a shaping device (e.g. plunger
stamp 660) can be used to recreate structure in an intestinal model
that mimic intestinal villi. Villi are understood to be a
predominant cellular structure of the in vivo intestine, as amongst
other things, they are believed to correspond to a villus-crypt
axis of cell differentiation. An ability to controllably form
structures that mimic villi in an intestinal model is another
beneficial embodiment of the disclosed open-top microfluidic
systems.
[0162] A type of pattern formed on a gel or porous volume may also
determine if desired cell types will form in, or on, the said gel
or porous volume. For example, adult keratinocyte cells may not
differentiate and may die if the geometry of the gel does not
sufficiently simulate the cells' native environment. Using a
patterned shaping device (e.g. patterned plunger stamp 660) that
allows the imprinting of specific and sophisticated patterns (e.g.,
patterning and/or geometries simulating the native environment for
cells being cultured) into the gel or porous volume surface, a
desirable micro-environment can be created that may allow for cell
survival and cell differentiation.
[0163] Turning now to FIG. 7, an exemplary pattern for a plunger
stamp 760 is illustrated. The plunger stamp 760 includes a
patterned bottom surface with a plurality of papillae structures
767 that simulate the papillae structure of the dermis, which when
imprinted into the surface of a gel layer can be useful for
differentiation of an adult skin equivalent.
[0164] In some embodiments, a gel layer is first placed into an
open region of a top structure of a microfluidic device or placed
into a mold (e.g., simulating an open region) followed by stamping
of a gel surface with a plunger stamp. In other embodiments, a
plunger stamp is first inserted into an open region to a
predetermined desired based on a desired gel layer thickness and a
pre-polymerized gel with a lower-viscosity than in its final cured
form is placed or allowed to flow into an open region confined by a
plunger stamp, a membrane, and the sides of the open region. A
plunger stamp is dimensioned such that there are sufficient
tolerances (e.g., gaps) between the side of a plunger stamp and the
side walls of an open region (e.g., channel) so that a gel does not
ooze or leak up the side of an open region when a pre-polymerized
gel is imprinted with a patterned surface of a plunger stamp.
[0165] Referring now to FIGS. 8-10, an exemplary embodiment of an
open-top device 800 including round open regions 804a, 804b, 804c
is illustrated. The round open regions 804a, 804b, 804c offer
advantages in the use of the device. For example, the device is
amenable to biopsy with round biopsy punches typical for in vivo
work, there is broad area available for topical treatments or
experimental procedures, and they may provide a more isotropic
biological environment than, for example, elongated sections. A
more isotropic environment can be especially beneficial when
present cells affect contractile or expansive forces, as is often
the case with fibroblasts such as those present in the dermal-like
layer of skin models. Although the depicted embodiments in FIG. 8
are round, some of the aforementioned advantages also apply to
other shapes, including for example ovals, shapes that inscribe
round sections, or other broad shapes.
[0166] FIGS. 8-10 specifically illustrate stretchable embodiments
of an open-top microfluidic device 800. A stretchable open-top
microfluidic device, such as the one illustrated in FIGS. 8-10 can
include open regions shaped in various ways including linear
sections, although circular, elliptical (e.g., from circular to a
1:2 ratio), or ovoid top region seem to reduce the impact of
tissue-induced stress that can lead to delamination of the tissue
culture of interest (e.g., skin tissues). A stretchable device may
allow for flow in a bottom fluidic layer that is separated from a
top fluidic layer by a permeable membrane (not shown), similar to
the open-top microfluidic devices described for FIGS. 3-5. While
the open-top microfluidic device 800 is described as a stretchable
device, it can be used with membranes other than stretchable
membranes (e.g., PDMS membranes) for applications where membrane
stretch is not desired.
[0167] Turning to FIG. 8A, a top view of the exemplary assembled
stretchable open-top microfluidic device 800 is illustrated. The
device 800 includes a top structure 820 that has three apertures
therethrough which define a plurality of open-top open regions
804a, 804b, 804c that may include a gel or porous volume. The
open-top open regions may extend through the entire thickness of
the top structure 820. As mentioned, mechanical actuation can be
effected in a variety of ways; in the illustrated example,
mechanical stretch is attained using one or more operating channels
that on the perimeter of the open region. The top structure 820
further includes a plurality of vacuum port pairs: i) 830a, 832a;
ii) 830b, 832b; and iii) 830c, 832c, that are in communication with
the one or more vacuum chambers 837a, 838a, 837b, 838b and 837c,
838c. The vacuum port pairs can be connected to a vacuum device
that is used to generate pressure differences that cause, for
example, a membrane (not shown) to stretch (e.g., radially). Each
open-top open region (e.g., 804a) is illustrated as having two
opposing vacuum ports (e.g., 830a, 832a), thereby forming a vacuum
port pair. The illustrated configuration permits a mechanical
stretch generated by the opposing vacuum chambers 837a-c and 838a-c
to apply a biaxial force on the device's membrane active regions.
Combined with the circular shape of the open regions, the device
approximates isotropic stretch, which may be desirable in the
recapitulation of the biological mechanical environment of some
organs, including the skin. In alternative embodiments, the shape
of the open regions and vacuum chambers can be modified to augment
the directionality and non-isotropicity of the stretch. Moreover,
devices that include a plurality of vacuum chambers corresponding
to one or more of the open regions allow the application of
different pressures (including vacuum levels) permitting the
selection of stretch directionality during use. The top structure
820 further includes a plurality of bottom fluidic layer inlet
ports 819a, 819b, 819c and outlet ports 822a, 822b, 822c that allow
for the introduction and extraction of fluids (e.g., for perfusion)
from the open-top microfluidic device 800. FIG. 8B illustrates a
perspective view of the top structure of the exemplary stretchable
open-top microfluidic device of FIG. 8A, and in particular shows
how the open-top open regions, vacuum ports, vacuum chambers and
bottom fluidic layer extend through the entire top structure 820.
More or fewer (e.g., one, two, four, five or more) open-top open
regions and related support features are contemplated.
[0168] Turning now to FIG. 8C, a perspective view of the bottom
structure 825 of the exemplary stretchable open-top microfluidic
device 800 is illustrated. Similar to the previously described
embodiments of an open-top microfluidic device, a permeable
membrane (not shown) is disposed along the interface between the
top structure 820 and the bottom structure 825. The bottom
structure includes feeding channels 839a-c comprising a plurality
of feeding channel wells (e.g., first feeding channel well 835a,
second feeding channel well 835b, third feeding channel well 835c)
that align with open-top inlet and outlet ports (e.g., 514 and
516), respectively. A membrane (not shown) separates the open-top
open region (e.g., 804a) from the feeding channels 839a-c and
feeding channel wells (e.g., 835a-c). It is contemplated that a gel
layer in the device 800 can be formed on top of the membrane in the
open-top openings similar to what is described elsewhere herein
(see, e.g., FIGS. 5C-5D).
[0169] FIGS. 9 and 10 illustrate exemplary perspective views of
cross-sections 9-9 and 10-10 through the stretchable open-top
microfluidic device. of FIG. 8A. With the top and bottom structure
assembled, the bottom fluidic layer inlet (e.g., 819b) and outlet
ports (e.g., 822b) each extend through the membrane (not shown)
such that the ports are each hydraulically connected to feeding
channels 839a, 839b, 839c (e.g., illustrated as long narrow
channels) in the bottom structure 825 to allow for the circulation
or introduction of fluids into the open-top microfluidic device.
FIG. 8. Similarly, in FIG. 9 the vacuum port pairs (e.g., 930a,
932a; 930b, 932b; 930c, 932c) in the top structure 920 each extend
to vacuum chamber pairs: i) 937a, 938a; ii) 937b, 938b; and iii)
937c, 938c formed by the interfacing of the top structure 920 and
bottom structure 925. The vacuum chambers are at least partially
defined by a stretchable or deformable surface pairs such as 1045b
and 1046b that introduces pressure changes to actuate the membranes
(not shown) at the interface of each of the open-top openings
(e.g., 1004b) and with the bottom wells (e.g., 1006b FIG. 10.
[0170] Although it is not necessary to understand the mechanism of
an invention, it is believed that the presently disclosed vacuum
chambers function to provide a pneumatic stretching of a membrane.
For example, when placed under a vacuum, a first deformable surface
1645 and second deformable surface 1646 deflect towards each other
as depicted by a deflection line 1647. FIG. 16. It is further
believed that since the top portion of the deformable surfaces are
deflected at a greater angle than the bottom portion of the
deformable surfaces, the induced stress is transferred to the
underlying membrane, thereby causing the membrane to stretch. A
more detailed depiction of deformable surfaces 1945, 1946 induce a
deflection 1947 that causes bending around the corner of the vacuum
chamber wall, as shown by the change in position of the inner and
outer dotted lines. FIG. 22.
[0171] FIGS. 11 and 12 illustrate exemplary views of different
bottom fluidic channel configurations. In the embodiment
illustrated in FIG. 11, a lower microchannel 1136 is split into a
number of constituent channels 1129. Although it is not necessary
to understand the mechanism of an invention, it is believed that
the smaller diameter of these constituent channels 1129, as
compared to the diameter of a lower microchannel 1136,may offer an
advantage in terms of bubble/debris clearance and flow uniformity
compared to the single wider channel. Alternatively, as illustrated
in FIG. 12, the lower microchannel 1236 can be take a spiral form
1251, or a serpentine or meandering form 2252 as illustrated in
FIG. 22. Although it is not necessary to understand the mechanism
of an invention, it is believed that the configuration of FIG. 12
can provide increased robustness in the face of bubbles and debris
that may be present, and can provide a more even flow rate than the
lower microchannel 1136 design illustrated in FIG. 11. However, the
resulting channel length of the lower microchannel 1236
configuration in FIG. 12 is typically longer than in the lower
microchannel 1136 designs similar to FIG. 11, with a shorter
microchannel length being advantageous in some applications. For
example, the spiral lower microchannel 1251 design illustrated in
FIG. 12 first winds inwardly towards the center of the active
region and the winds outwardly. An alternative design avoids the
outward winding by flowing downward, either to a fluidic port or to
an additional fluidic channel that may run underneath the spiral
channel.
[0172] In one embodiment, the present invention contemplates an
open-top microfluidic device 1300 comprising at least two open
regions 1304. Each open region 11304 may be configured with an
inlet port 1314, an outlet port 1316 and a vacuum port pair (1330,
1332).
[0173] In one embodiment, the present invention contemplates an
open top microfluidic device 1400 comprising a top chamber 1407 or
a bottom chamber 1406, said chambers having side walls 1443 where a
plurality of projections 1413 protrude into a chamber lumen 1421.
FIG. 14A and FIG. 14B.
[0174] In one embodiment, the present invention contemplates an
open-top chip device 1500 comprising at least two spiral lower
microchannels 1551, wherein each of the microchannels are in
fluidic communication with an inlet port 1519 and an outlet port
1522. FIG. 15.
[0175] In one embodiment, the present invention contemplates an
open-top chip device 1700 comprising: i) a first chamber 1763 and a
second chamber 1764, wherein each chamber is surrounded by a
deformable surface 1745; and ii) at least two spiral microchannels
1751 located on the bottom surface of the chambers, wherein each of
the microchannels are in fluidic communication with an inlet port
1719 and an outlet port 1722 and are respectively configured with a
first vacuum port 1730 or a second vacuum port 1732, such that each
vacuum port is respectively connected to a first vacuum chamber
1737 or a second vacuum chamber 1738. FIG. 17. An exploded view of
the embodiment depicted FIG. 17 shows an open-top chip device 1800,
wherein a membrane 1840 resides between the bottom surface of the
first chamber 1863 and the second chamber 1864 and the at least two
spiral microchannels 1851. FIG. 18.
[0176] In some embodiments, the present invention contemplates an
open-top chip device 2700 comprising at least two spiral lower
microchannels 2751, wherein the microchannels are in fluidic
communication with an inlet port 2719 and an outlet port 2722. FIG.
27. The spiral lower microchannel 2751 is also flanked by a vaccum
port 2730 configured with a vacuum chamber 2737. A deformable
surface 2745 is configured on the inside surface of the vacuum
chamber 2737,
[0177] In some embodiments, the present invention contemplates an
array device 2811 comprising a plurality of open top chip devices
2800. Each of the open top chip devices 2800 is configured with at
least an open region 2804 and flanked by an inlet port 2814 and an
outlet port 2816, or alternatively, a first vacuum port 2730 and a
second vacuum port 2732. FIG. 28. An exploded view of an array
device 3911 is provided showing the open top chip devices 3900 in
top structure 3920 and a bottom chamber 3906 in bottom structure
3925 with a membrane 3940 layered between the top structure 3920
and bottom structure 3925. FIG. 39.
[0178] Although it is not necessary to understand the mechanism of
an invention, it is believed that an array device comprising open
top chips represents a fundamental shift in architecture as
compared to conventional "tissue-on-a-chip" designs. It is further
believed that this array design facilitates multiplexing of 3D
scaffold models for scaffold optimization. Furthermore, the array
test platforms are designed to be compatible with existing 3D
scaffold models in transwell. For example, array devices as
contemplated herein are useful for 3D scaffold models, ECM/gel
optimization and tissue chips including, but not limited to, skin,
lung and intestine (e.g., gut). In one embodiment, an array device
2811 may have the following specifications:
TABLE-US-00001 Body Material PDMS Sylgard 184 Membrane Material
PDMS Sylgard 184 Dimensions Width 51.8 mm Length 50.8 mm Height 8.0
mm Open-Top Chamber Dimensions Top Chamber Diameter 6.3 mm Top
Chamber Height 6 mm Top Channel Volume 193.02 mm.sup.3 Top Culture
Area 32.17 mm.sup.2 Bottom Chamber Dimensions Bottom Chamber
Diameter 5.4 mm Bottom Channel Height 1 mm Bottom Channel Volume
22.90 mm.sup.3 Bottom Culture Area 22.90 mm.sup.2 Membrane
Dimensions Pore Diameter 7 .mu.m Pore Spacing 40 .mu.m (hexagonally
packed) Thickness 50 .mu.m Co-culture Area 22.9 mm.sup.2 Minimum
Imaging Distance (top of membrane) 2 mm
[0179] Additional exemplary embodiments of open-top microfluidic
devices, such as the devices discussed above in FIGS. 1-12, are now
described further. In some embodiments, the dimensions of the top
area of the open region in a top structure for a chip can range
from about 0.1 to about 17 millimeters (or 1 to about 7
millimeters) along in the narrowest dimension. In some embodiments,
the dimensions range from about 0.5 to about 200 or more
millimeters (or about 0.5 to about 20 millimeters). The lower end
of the range of the narrowest dimension of the open region is also
desirably sized to allow accessibility to the region for pipettes
or syringes that are used to place, for example cell cultures or
gel materials. The open region can be sized to limit any capillary
action, which may be undesirable in some applications (capillary
action may nevertheless be desirable in other applications). It is
further desirable in some applications for the upper range of the
open region dimensions to be sized to maintain accuracy in the flow
distribution for the bottom channel across the cell culture
area.
[0180] In some embodiments, the depth of the open region (e.g.,
measuring vertically upward in the open region from the interface
of the top structure with the membrane) can vary from about 0.1 to
about 20 millimeters (or about 1 to about 5 millimeters). In some
embodiments, an additional well or spacer may be added to increase
the well volume of the open region, such as where the full depth of
the open region is completely filled. It is contemplated that
aspect ratios of the dimensions for the top area to the depth of
the open region in some applications should range from about 1 to
above 100, or in some applications from about less than 0.01 to
2.
[0181] In some embodiments, it is desirable to have different
geometries for the open region based on the type of tissue that is
subject to experimentation. For example, certain types of tissue,
such as skin, are highly contractile during culturing. When placed
into high-aspect ratio (e.g., 16 millimeters by 1 millimeter)
channels, delamination of the tissue can occur along the narrow
dimension. However, an open region that has a circular (e.g., open
region 804a) provides radial symmetry that can allow tissue to
shrink uniformly and not move out of plane. A wider channel
geometry that minimizes edge effects can also be beneficial for
other organ systems that may require multiple layers, such as the
blood-brain barrier, airways, or digestive tract, because the
layers can be more easily formed by the sequential deposition of
thin gel or cellular tissue layers, which is difficult to do in
closed channels or chambers. In some embodiments, the geometry of
the open region is something different than the rectangles or
circles illustrated in the exemplary embodiments of FIGS. 5 and 8.
For example, a triangular or star geometry can be used to look at
the effects of cell crowding or diffusion of signaling molecules as
affected by geometry. In another example, a "FIG.-8" shape can be
beneficial for analyzing the interaction between two
three-dimensional cultures
[0182] For fluidic channel(s) disposed in the top structure of an
open-top device that might be used for skin, bronchial, or gut
tissue simulations, the geometry and dimensions for the open region
of a chamber can include a channel-type geometry with a channel
height ranging, for example, from about 0.02 millimeters to about
10 millimeters, a channel width of about 0.05 millimeter to 20
millimeter, and a channel length of about 0.5 millimeters to about
300 millimeters. In some embodiments, the geometry and dimensions
for the open region of a top chamber can include a channel-type
shape with a height ranging, for example, from about 0.02
millimeters to about 10 millimeter and a fluidic cover fluidic
channel width of about 0.05 millimeter to 20 millimeter. The base
or bottom chamber can also have a channel-type shape with a height
ranging, for example, from about 0.02 millimeters to about 10
millimeter. For an optional top structure 420 that might be used
for brain-barrier and lung tissue simulations, the geometry and
dimensions for the top structure, for example, include a height of
about 0.05 millimeters to about 5 millimeter. A taller top
structure spacer in an open-top microfluidic device is often used
for simulations where three-dimensionality is desirable, such as
where fibroblast or other cells are embedded in the gel layer for
the formation of, for example, a dermal layer. A shorter top
structure spacer in an open-top microfluidic device can be used,
for example, for simulations where two- or three-dimensionality is
desired, such as for small airway simulations where small airway
cells feel the paracrine stimulation of neighbor cells, which
stimulates their full differentiation.
[0183] Various tissue types are contemplated for testing in an
open-top microfluidic device (e.g., an open-top OOC device), such
as skin, small-airway, and alveolar tissues. However, open-top
microfluidic devices can also accommodate other types of tissues,
as well, including other epithelial tissues.
[0184] The properties of gels or porous volumes that can be used
for an open-top microfluidic device can vary and the properties
will often depend on the different tissue type that is being
tested. For example, different tissue types or specific models may
employ different extracellular matrix proteins (ECMs) and ECM
mixtures (for example, collagen I, collagen IV, Matrigel.RTM.,
laminin, fibronectin, gelatin, elastin, etc., and combinations
thereof). Additionally, some embodiments may employ synthetic
polymer gels (e.g. polyacrylamide, polyvinyl alcohol, etc.) or
various other gels known in the art (e.g. agarose, alginate, etc.)
alone, in mixture, or in combinations with ECMs. Similarly, porous
volumes used for an open-top microfluidic device may include a
variety of open-cell foams, for example, expanded polyurethane,
expanded polystyrene, expanded cellulose, expanded polylactic acid,
etc. Without being bound by example, for the simulation of a skin
or bronchial tissue, the gel can have a higher concentration of
collagen, roughly at about 1 to about 11 milligrams per milliliter
of gel. For the simulation of gut tissue function, one exemplary
embodiment contemplates a gel with a 1:1 ratio of a high
concentration collagen to an ECM such as the Corning.RTM.
Matrigel.RTM. matrix available from Corning Life Sciences, is
desirable. For the simulation of alveolar tissue function, one
exemplary embodiment contemplates a gel with a 1:1 ratio of a low
concentration of collagen (e.g., about 3 milligrams per milliliter
of gel) to ECM, such as the Corning.RTM. Matrigel.RTM. matrix or
fibronectin, is desirable. It is contemplated in one embodiment
that extracellular matrices or other gel precursors that form gels
with concentrations of above 5 milligrams per milliliter of gel, or
ranging from about 3 to about 15 milligrams per milliliter of gel,
or ranging from about 0.2 to 4 milligrams, can be used in the
open-top microfluidic devices described herein. Moreover,
cross-linking agents such as, but not limited to, transglutaminase,
glutaraldehyde, bis(sulfosuccinimidyl)suberate, and many other
cross-linkers known in the art, can be used to increase gel
stiffness and optionally lower gel concentration. With the use of
cross-linkers, it is contemplated that extracellular matrices or
other gel precursors that form gels with concentrations ranging
from about 0.05 to 5 milligrams per milliliter of gel, or ranging
from about 1 to 10 milligrams per milliliter of gel, can be used in
the open-top microfluidic devices described herein.
[0185] While the described open-top microfluidic devices, including
open-top OOC devices, are compatible with standard microfluidic
fluids having relatively low viscosities (e.g., about 1 to about 10
centipoise or less), the open-top devices are well-suited for high
viscosity solutions and gels having a viscosity equal to or greater
than 10 centipoise along with being well-suited for the
polymerization of gels in situ for later removal from the
microfluidic device and other manipulation of the gel. For example,
collagen gels with a high protein content (e.g., 3 milligrams per
milliliter) can be directly pipetted into the open tops and gelled
in place without shearing cells or requiring high pressure
actuation. For drug testing applications, creams and similar
high-viscosity materials can be spread directly on the tissue using
the open tops to test compounds in the final formulations rather
than dissolved drugs alone. Thick gels layers can also be easily
generated for three-dimensional culture applications with the
potential for providing mechanical stretch. Other desirable
embodiments of open-top microfluidic devices include the open tops
are readily compatible with aerosol and other particulate (e.g.,
liquid or solid) delivery while minimizing loss, which allows for
enabling high dosing accuracy. Because the particles can be
delivered directly to the tissue, there is minimal loss due to
adsorption to other surfaces, such as tubing and microchannels.
[0186] In some embodiments, the gel layer described in the above
embodiments does not need to be patterned. It is also contemplated
that a gel or other material suitable for growing tissues can be
patterned externally, shaped to fit the open region of the channel
or chamber of the top structure, and subsequently inserted into the
open-top microfluidic device for cell culture. The gel or other
material could also be a large sheet that is compressed using the
spring loaded clamps with the two chambers or channels on either
side of the gel or other material, where the gel or other material
acts as a membrane in the open-top microfluidic device. The
externally-prepared material can include biological tissue such as
a biopsy from a patient or small piece of artificial tissue prior
to implantation, and thus allow the performance of assays on tissue
to determine drug response, tissue quality, and other factors. It
is further contemplated that the gel or a similar material from the
open-top microfluidic device can be extracted via the open top and
used for in vivo applications. For example, the microfluidic device
could be used to pattern and mature the tissue prior to
implantation.
[0187] Numerous skin substitutes are commercially available, such
as epidermal substitutes, dermal substitutes, and bilayer
substitutes. These can be employed together with the devices,
layered structures and methods described above.
Preferred Embodiments
[0188] A. Blood Brain Barrier
[0189] Brain microvascular endothelial cells (BMEC) are
interconnected by specific junctional proteins forming a highly
regulated barrier separating blood and the central nervous system
(CNS), the so-called blood-brain-barrier (BBB). Together with other
cell-types such as astrocytes or pericytes, they form the
neurovascular unit (NVU), which specifically regulates the
interchange of fluids, molecules and cells between the peripheral
blood and the CNS.
[0190] The blood-brain barrier is of major clinical relevance
because dysfunction of the blood-brain barrier leads to
degeneration of the neurovascular unit, and also because drugs that
are supposed to treat neurological disorders often fail to permeate
the blood-brain barrier. Due to its importance in disease and
medical treatment, it would be highly advantageous to have a
predictive model of the human blood-brain barrier that
recapitulates aspects of the cerebral endothelial microenvironment
in a controlled way.
[0191] In one embodiment, the present invention contemplates a
layered structure comprising i) fluidic channels covered by ii) a
porous membrane, said membrane comprising iii) a layer of brain
microvascular endothelial cells and said membrane positioned below
iv) a gel matrix (or other porous volume). The present invention
contemplates, in one embodiment, living neuronal cells (e.g.
neurons, astrocytes, pericytes, etc.) on, in or under the gel
matrix. It is preferred that some portion of the device can be
opened for access to these cells. In one embodiment, the device
comprises a removable top. The gel can be patterned to control the
positioning and/or orientation of the cells or portions thereof.
For example, the pattern on the gel matrix can direct neurite
growth for neurons seeded on the patterned surface.
[0192] B. Transepithelial Electric Resistance
[0193] There are many ways to evaluate the integrity and physiology
of an in vitro system that mimics the blood brain barrier. Two of
the most common methods are Transepithelial Electric Resistance
(TEER) and Lucifer Yellow (LY) rejection. Lucifer Yellow (LY)
travels across cell monolayers only through passive paracellular
diffusion (through spaces between cells) and has low permeability.
Therefore it is considerably impeded in passing across cell
monolayers with tight junctions. Permeability (Papp) for LY of
.ltoreq.5 to 12 nm/s has been reported to be indicative of
well-established monolayers. One of skill in the art would
understand that manipulations should be performed using aseptic
techniques in order for the cells to remain in culture without
contamination. TEER measures the resistance to pass current across
one or more cell layers on a membrane. Specifically, this
electrical resistance is a direct measurement of the resistance of
the cell monolayer to the transport of ions. The measurement may be
affected by the pore size and density of the membrane, but it aims
to ascertain cell and/or tissue properties. The TEER value is
considered a good measure of the integrity of the cell
monolayer.
[0194] For TEER measurements, an embodiment is contemplated wherein
a layered structure or microfluidic device 2300 has a top electrode
2371 and a bottom electtrode 2372 configured for measuring the
electrophysiology of said brain microvascular endothelial cells.
FIG. 23 In one embodiment, the top electrode 2371 is a
chromium/gold (Cr--Au) electrode. In one embodiment, the bottom
electrode 2372 is a chromium/gold (Cr--Au) electrode.
[0195] However, it is not intended that the present invention be
limited to only TEER measurements. In one embodiment, the present
invention contemplates a method of testing, comprising 1) providing
a layered TEER microfluidic device 2300 comprising i) a bottom
structure 2325 comprising at least one upper microfluidic channel
2334 covered by ii) a porous membrane 2340, said membrane
comprising iii) a layer of brain microvascular endothelial cells in
contact with said at least one upper microfluidic channel, said
membrane position below iv) a gel matrix (or other porous volume),
said gel matrix (preferably) under a removable cover; and 2)
measuring the electrophysiology of said brain microvascular
endothelial cells. In one embodiment, the porous membrane 2340 is
covered by a top structure 2320. In one embodiment, the layered
TEER microfluidic device 2300 further comprises a top clamp 2379
and a bottom clamp 2384, wherein said top clamp 2379 has at least
one access hole 2381. In one embodiment, the at least one access
hole 2381 is configured to align with a port adapter 2383. In some
embodiments, a glass slide 2382 is placed between the bottom
electrode 2372 and the bottom clamp 2384. In one embodiment, the
top clamp 2379 comprises a lasercut acrylic material. In one
embodiment, the port adapter 2383 comprises a cast PDMS material.
In one embodiment, the top electrode 2371 comprises a lasercut PET
material. In one embodiment, the bottom electrode 2372 comprises a
lasercut PET material. In one embodiment, the top structure 2320
comprises an open-top channel gasket having a cast PDMS material.
In one embodiment, the bottom structure 2325 comprises an
open-bottom channel gasket having a spincoated and lasercut PDMS
material. In one embodiment, the bottom clamp 2384 comprises a 3D
printed ABS plastic material. Although not limiting, the top clamp
2379 and bottom clamp 2384 may be attached with M4 screws 2386 and
M4 nuts 2387. Although it is not necessary to understand the
mechanism of an invention, it is believed that a TEER microfluidic
device is clamped because the various layered components described
above would be difficult to glue (e.g., bonding). It is further
believed that a clamp facilitates an ability to open the device and
have direct access to cells for patch-clamp measurements.
Alternatively, if this openable feature is not desired, the device
layers can be bonded together. A fully assembled layered TEER chip
2400 between a top clamp 479 and bottom clamp 2384 is presented in
FIG. 24.
[0196] A variety of techniques are contemplated including but not
limited to using a multi-electrode array or patch clamping. In one
embodiment, the present invention contemplates an "open top" design
that allows for patch clamping through the opening. For example, an
open-top patch clamp layered TEER microfluidic device 2500 may
comprise an optional top microfluidic cover 2510 comprising an open
region 2504, an optional top microfluidic cover fluidic channel
2508 and inlet port 2514, wherein the open region 2504 provides
access to an open-top channel gasket 2573. In one embodiment, the
TEER microfluidic subassembly device 2500 comprises an open-top
channel gasket 2573 having at least one upper microchannel 2534 in
fluid communication with at least one upper microchannel well 2523.
A porous membrane 2540 is placed between the open-top channel
gasket 2573 and an open-bottom channel gasket 2574, wherein the
open-bottom channel gasket 2574 comprises at least one lower
microchannel 2536. A bottom electrode 2572 is placed underneath the
open-top channel gasket/porous membrane/open-bottom channel gasket
layered stack. In one embodiment, the bottom electrode 2572 is a
chromium/gold electrode. FIG. 25
[0197] An open-top TEER microfluidic subassembly patch clamp device
2600 may be exposed to allow access with a micro-manipulator 2661.
FIG. 26. For example, a micromanipulator arm 2661 my be placed
directly within an upper microchannel 2634. Although it is not
necessary to understand the mechanism of an invention, it is
believed that the micromanipulator arm 2661 may, for example, add
reagents, remove a fluid sample, add cells and/or remove cells.
This allows the configuration of the patch clamp device 2600 to
interchangeably go between a flow configuration (e.g., where the
upper microchannel 2634 is not exposed) and an open configuration
(e.g., where the upper microchannel 2634 is exposed).
[0198] C. Stretchable Open Top Chips
[0199] In one embodiment, the present invention contemplates a
stretchable open top chip device 2900 comprising at least one
spiral microchannel 2951 configured with at least one fluid inlet
2917 and at least one fluid outlet 2924. FIG. 29A. In one
embodiment, the microfluidic chip device 2900 further comprises a
upper microchannel with a circular chamber 2956 configured with a
first fluid or gas port pair 2975 and second fluid or gas port pair
2976, a first vacuum port 2930 connected to a first vacuum chamber
2937 and a second vacuum port 2932 connected to a second vacuum
chamber 2938, wherein the vacuum chambers are proximally configured
around the spiral microchannel. In one embodiment, the upper
microchannel with a circular chamber 2956 is positioned above the
spiral microchannel 2951. FIG. 29B.
[0200] Although it is not necessary to understand the mechanism of
an invention it is believed that the strechable open top chip
design represents a fundamental shift in architecture as compared
to conventional "tissue-on-a-chip" designs. It is further believed
that the open top design is compatible with 3D scaffold models. For
example, an open top chip design may include, but is not limited
to, three layers exemplified by a bottom channel, a middle chamber
and a top channel. In one embodiment, the bottom channel layout may
be spiral in shape in order to fit within the circular shape of the
chamber. In another embodiment, the top channel allows for the
ability to run media solutions or humidity-controlled gases (e.g.,
for example, air and/or oxygen-carbon dioxide mixtures such as 95%
O.sub.2/5% CO.sub.2) to prevent gel evaporation. In another
embodiment, the membrane is porous to facilitate cell-to-cell
communication. Other embodiments provide a vacuum channel design
that provides a mechanical stretch to the entire 3D scaffold
thickness.
[0201] Furthermore, the open top strechable chips as contemplated
herein are useful for biological interfaces, co-cultures, multiple
cell type cultures, tissue streching, 3D scaffold models,
micro-patterning and tissue chips including, but not limited to,
skin, lung and intestine (e.g., gut). In one embodiment, an open
top strechable device may have the following specifications:
TABLE-US-00002 Body Material PDMS Sylgard 184 Membrane Material
PDMS Sylgard 184 Dimensions Width 15.87 mm Length 35.87 mm Height
6.0 mm Top Channel Dimensions Top Channel Height 200 .mu.m Top
Chamber Diameter 5.70 mm Top Chamber Dimensions Top Chamber
Diameter 5.70 mm Top Chamber Height 4.00 mm Top Channel Volume
102.07 mm.sup.3 Top Culture Area 25.52 mm.sup.2 Bottom Channel
Dimensions Bottom Channel Width 600 .mu.m Bottom Channel Height 400
.mu.m Bottom Channel Volume 5.446 mm.sup.3 Bottom Culture Area 13.6
mm.sup.2 Membrane Dimensions Pore Diameter 7.0 .mu.m Pore Spacing
40 .mu.m (hexagonally packed) Thickness 50 .mu.m Minimum Imaging
Distance (top of membrane) 850 mm
[0202] In one embodiment, the present invention contemplates a
stretchable open top chip device 3000 comprising: i) a fluidic
cover 3010 comprising an upper microchannel with a circular chamber
3056 configured with a first fluid or gas port pair 3075 and second
fluid or gas port pair 3076; a fluid inlet port 3014, a fluid
outlet port 3016, a first vacuum port 3030 and a second vacuum port
3032; ii) a top structure 3020 comprising a chamber 3063, a first
vacuum chamber 3037 connected to the first vacuum port 3030, and a
second vacuum chamber 3038, connected to the second vacuum port
3032, wherein the upper microchannel with a circular chamber 3056
overlays the top surface of the chamber 3063; and iii) a bottom
structure 3025 comprising a spiral microchannel 3051 comprising an
inlet well 3068 connected to the fluid inlet port 3014 and an
outlet well 3069 connected to the fluid outlet port 3016, wherein a
membrane 3040 is layered between the top struture 3020 and bottom
structure 3025. FIG. 30.
[0203] In one embodiment, the present invention contemplates a
stretchable open top chip device 3100 comprising a chamber 3163
comprising an epithelial region 3177 and a dermal region 3178. In
one embodiment, the epithelial region comprises an epithelial cell
layer. In one embodiment, the dermal region comprises a dermal cell
layer, wherein said epithelial cell layer adheres to the surface of
the dermal cell layer. In one embodiment, the device further
comprises a spiral microchannel 3151 in fluid communication with a
fluid inlet port 3114, wherein the microchannel comprises a
plurality of vascular cells. In one embodiment, a membrane 3140 is
placed between the chamber dermal cell layer and the microchannel
plurality of vascular cells. In one embodiment, the device further
comprises an upper microchannel with a circular chamber 3156
connected to a fluid or gas port pair 3175. In one embodiment, the
device further comprises a first vacuum port 3130 connected to a
first vacuum chamber 3137 and a second vacuum port 3132 connected
to a second vacuum chamber 3138. In one embodiment, the membrane
3140 comprises a PDMS membrane comprising a plurality of pores
3141, wherein said pores 3141 are approximately 50 .mu.m thick,
approximately 7 .mu.m in diameter, packed as 40 .mu.m hexagons,
wherein each pore has a surface area of approximately 0.32
cm.sup.2. Although it is not necessary to understand the mechanism
of an invention, it is believed that the pore surface area contacts
a gel layer (if present). FIGS. 31A and 31B.
[0204] In one embodiment, the present invention contemplates a
stretchable open top chip device 3200 comprising: i) a fluidic
cover 3210 comprising an upper microchannel with a circular chamber
3256 configured with a first fluid or gas port pair 3275 and second
fluid or gas port pair 3276; a fluid inlet port 3214, a fluid
outlet port 3216, a first vacuum port 3230 and a second vacuum port
3232; ii) a top structure 3220 comprising a chamber 3263, a first
vacuum chamber 3237 connected to the first vacuum port 3230, and a
second vacuum chamber 3238, connected to the second vacuum port
3232, wherein the upper microchannel with a circular chamber 3256
seals with the top surface of the chamber 3263; and iii) a bottom
structure 3225 layered underneath said top structure 3220. FIG.
32.
[0205] FIGS. 33A and 33B illustrate exploded views of two
embodiments of a stretchable open top chip device comprising: i) a
fluidic cover 3310 comprising an upper microchannel with a circular
chamber 3356 configured with a first fluid or gas port pair 3375
and second fluid or gas port pair 3376; a fluid inlet port 3314, a
fluid outlet port 3316, a first vacuum port 3330 and a second
vacuum port 3332; ii) a top structure 3320 comprising a chamber
3363, a first vacuum chamber 3337 connected to the first vacuum
port 3330, and a second vacuum chamber 3338, connected to the
second vacuum port 3332, wherein the upper microchannel with a
circular chamber 3356 overlays the top surface of the chamber 3363
and a first membrane 3340 layered between the fluidic cover 3310
and the top structure 3320; and iii) a bottom structure 3325
layered underneath said top structure 3220, wherein a second
membrane 3340 is layered between the bottom structure 3325 and the
top structure 3320. FIG. 33.
[0206] FIG. 34A illustrates an assembled top view of a stretchable
open top chip device as shown in FIG. 33A. FIG. 34B illustrates a
cutaway assembled side view of a stretchable open top chip device
as shown in FIG. 33A.
[0207] In one embodiment, the present invention contemplates a tall
channel stretchable open top chip device 3500 comprising: i) a
fluidic cover 3510 comprising an open region 3504; ii) a top
structure 3520 comprising an upper microchannel 3534 attached to
the fluidic cover 3510; iii) a bottom structure 3525 comprising a
lower microchannel 3536 attached to the top structure 3520; and iv)
a membrane 3540 layer between the bottom structure 3525 and the top
structure 3520. In one embodiment, the open region 3504, upper
microchannel 3534 and lower microchannel 3536 are configured to at
least partially overlay each other. FIG. 35A and FIG. 35B. Although
not intended to be limiting, the tall channel stretchable open top
chip device 3500 may also comprise a vacuum port pair and/or
inlet/outlet ports as shown and described above.
[0208] Although it is not necessary to understand the mechanism of
an invention it is believed that a tall channel strechable open top
chip design represents a fundamental shift in architecture as
compared to conventional "tissue-on-a-chip" designs. It is further
believed that this tall channel open top design incorporates an
openable lid for direct access to the top channel that allows for
the ability to load thick gel matricies as well as micro-patterning
of the gel.
[0209] Furthermore, the open top strechable test platforms as
contemplated herein are useful for biological interfaces,
co-cultures, multiple cell type cultures, tissue streching, 3D
scaffold models, micro-patterning and tissue chips including, but
not limited to, skin, lung and intestine (e.g., gut). In one
embodiment, a tall channel open top strechable device may have the
following specifications:
TABLE-US-00003 Body Material PDMS Sylgard 184 Membrane Material
PDMS Sylgard 184 Dimensions Width 15.87 mm Length 35.87 mm Height
5.85 mm Top Channel Dimensions Top Channel Width 1000 .mu.m Top
Channel Height (closed) 1000 .mu.m Top Channel Height (open) 2000
.mu.m Top ChannelVolume 28.041 mm.sup.3 Top Culture Area 28.0
mm.sup.2 Bottom Channel Dimensions Bottom Channel Width 1000 .mu.m
Bottom Channel Height 200 .mu.m Bottom Channel Volume 5.584
mm.sup.3 Bottom Culture Area 24.5 mm.sup.2 Membrane Dimensions Pore
Diameter 7.0 .mu.m Pore Spacing 40 .mu.m (hexagonally packed)
Thickness 50 .mu.m Co-culture Area 17.1 mm.sup.2 Minimum Imaging
Distance (top of membrane) 850 mm
[0210] In one embodiment, the present invention contemplates a
fully assembled stretchable open top microfluidic device 3600
comprising a fluidic cover 3610 comprising microfluidic channel
3608, a first vacuum port 3630 and a second vacuum port 3632,
wherein the microfluidic channel 3608 terminates at either end an
an inlet port 3614 and an outlet port 3616, respectively. FIG.
36.
[0211] A first cross-sectional view across plane A of FIG. 36
presents an open top microfluidic device 3700 in an assembled
configuration comprising a fluidic cover 3710 attached to a
membrane 3740, wherein the membrane 3740 overlays an open region
3704 (shown as hidden open region 3604 in FIG. 36) within a top
structure 3720 that is attached to a bottom structure 3725. FIG.
37A. A second cross-section view across plane A of FIG. 36 presents
an open top microfluidic device 3700 in a separated configuration
where a fluidic top 3710 comprising a membrane 3740 is removed from
top structure 3720 thereby providing access to an open region 3704,
wherein a microfluidic channel 3608 is configured within the
fluidic cover 3710. FIG. 37B.
[0212] A third cross-sectional view across plane A of FIG. 36
presents an open top microfluidic device 3800 in an assembled
configuration comprising a fluidic cover 3810 attached to a
membrane 3840, wherein the membrane 3840 overlays an open region
3804 (shown as hidden open region 3604 in FIG. 36) within a top
structure 3820 that is attached to a bottom structure 3825. FIG.
38A. A fourth cross-section view across plane A of FIG. 36 presents
an open top microfluidic device 3800 in a separated configuration
where a fluidic top 3810 comprising a membrane 3840 is removed from
top structure 3820 thereby providing access to an open region 3804,
wherein a microfluidic channel 3608 is configured to traverse
between fluidic cover 3810 and top structure 3820. FIG. 38B.
EXPERIMENTAL
Example 1
Keratinocyte and Fibroblast Cell Culture
[0213] This example describes the preparation of keratinocytes, and
in particular human foreskin keratinocytes (HFKs). An aliquot of
Lonza Gold KGM media (Lonza 192060) is placed in a 50 ml tube (i.e.
with 1 cryovial of HFK cells, one needs 12 ml for the flask, 10 ml
for the washing step and 1 to 5 ml to break the pellet for a total
of about 25 ml). The medium is warmed by putting it into the water
bath for 5-10 min and then transferred inside the sterile hood. The
needed number of 15 and 50 ml conical tubes are prepared, along
with the needed number of flasks. These are filled with the
appropriate amount of Lonza medium.
[0214] To thaw the HFKs, a cryovial is removed from the liquid
nitrogen container and transferred into the basket containing dry
ice. The cryovial is placed into the water bath until the freezing
medium inside it is completely melted. The cryovial is sprayed with
ethanol and brought to the sterile hood. The cryovial is opened in
the hood and the contents are collected from the cryovial (freezing
medium+cells) using a 1000 .mu.l pipette. The contents are
transferred into the 15 ml conical tube containing Lonza Gold KGM
medium previously warmed. This conical tube is closed and then
tilted to mix. Thereafter, it is centrifuged at 1000 rpm for 5
minutes. The conical tube is sprayed with ethanol and returned to
the sterile hood. It is opened and the supernatant is withdrawn,
leaving the cell pellet. The pellet is re-suspended using fresh
pre-warmed Lonza Gold KGM and the mixture is transferred to a flask
(or flasks), which were previously filled with Lonza Gold KGM
medium. The flasks are gently agitated to make sure that the medium
covers the entire bottom surface. The flasks are then transferred
to the incubator. The keratinocytes are fed with new media
approximately every other day (about every 36 hours).
[0215] To thaw the fibroblasts, a cryovial is removed from the
liquid nitrogen tank and transferred into the basket containing dry
ice. The cryovial is placed into the water bath until the freezing
medium inside it is completely melted. The cryovial is sprayed with
ethanol and brought to the sterile hood. The cryovial is opened in
the hood and the contents are collected from the cryovial (freezing
medium+cells) using a 1000 .mu.l pipette. Tee contents are
transferred into the 15 ml conical tube containing Lonza FGM-2
medium previously warmed. This conical tube is closed and then
tilted to mix. Thereafter, it is centrifuged at 1200 rpm for 5
minutes. The conical tube is sprayed with ethanol and returned to
the sterile hood. It is opened and the supernatant is withdrawn,
leaving the cell pellet. The pellet is re-suspended using fresh
pre-warmed Lonza FGM-2 and the mixture is transferred to a flask
(or flasks) which were previously filled with Lonza FGM-2 medium.
The flasks are gently agitated to make sure that the medium covers
the entire bottom surface. The flasks are then transferred to the
incubator. The fibroblasts are fed with new media approximately
every other day (about every 36 hours).
[0216] For detaching the HFKs by trypsinization, the protocol is as
follows. First, an aliquot Lonza Gold KGM (Lonza 192060), Lonza
reagent subculture reagent CC-5034 and E-medium (or variants) 10%
FBS medium is placed in 15 ml and 50 ml tubes. It is convenient to
us 4 mls of Lonza reagent subculture reagent CC-5034 per T75 flask
and to add 8 mls of 10% FBS medium to the flask (which corresponds
to 2 ml for each ml of reagent Lonza reagent subculture reagent
CC-5034). The media and enzymes are warmed by putting it into the
water bath for 5-10 min. The flask containing HFK (typically when
the cells are between 50 and 70% confluence) is removed from the
incubator, sterilized on the outside with ethanol, and transferred
into the hood. The flask is opened and the the Lonza Gold KGM
medium is aspirated, being careful to not scratch the bottom flask
surface where the cells are attached. Fresh pre-warmed Lonza Gold
KGM medium (e.g. 5 mls) is then added to wash the cells. This media
is also aspirated carefully. Then, 4 ml of 0.05% trypsin/EDTA
(Corning 25-052 CL) is added to the flask and the flask is returned
to the incubator. The detaching cells can be monitored using the
microscope if desired. As a rule of thumb, keratinocytes should
detach in about 2-3 minutes. Longer exposure to Lonza subculture
reagent CC-5034 (or 0.05 EDTA trypsin Invitrogen 25200-056) could
damage keratinocytes irreversibly. When the cells detach
completely, the outside of the flask is sterilized and brought to
the hood. The flask is opened and 8 ml of 10% FBS E-medium (or
variants) is added to the flask (2 ml for each ml of 0.05 EDTA
trypsin Corning 25-052-CL). Thereafter, the contents of the flask
are conveniently transferred to a 15 ml conical tube. The tube is
closed and centrifuged at 1000 rpm for 5 min. The tube is then
sterilized with ethanol, returned to the hood and opened. The
supernatant is gently aspirated, being careful not to disturb the
cell pellet. After the supernatant is removed, the pellet is
re-suspended using fresh pre-warmed Lonza Gold KGM medium. The
mixture is then transferred to the flask/flasks, which were
previously filled with Lonza Gold KGM medium. The flasks are gently
agitated to make sure that the medium covers the entire bottom
surface, and they are returned to the incubator. Feeding is as
stated above.
[0217] For detaching the fibroblasts by trypsinization, the
protocol is as follows. An aliquot of Lonza FGM-2 medium (Lonza
CC-3132), Lonza reagent subculture reagent CC-5034 and 10% FBS
medium is added in 15 ml and 50 ml tubes. It is convenient to use 4
ml Lonza reagent subculture reagent CC-5034 per T75 flask and 8 ml
of 10% FBS medium to the flask (which corresponds 2 ml for each ml
of reagent Lonza reagent subculture reagent CC-5034). The media and
enzymes are warmed by putting them into the water bath for 5-10
min. The flask containing fibroblasts (typically when the cells are
between 50 and 70% confluence) is removed from the incubator,
sterilized on the outside with ethanol, and transferred into the
hood. The flask is opened and the media is aspirated gently, being
careful to not scratch the bottom flask surface containing the cell
layer. 5 ml of fresh PBS is added to wash the cells (this can be
done twice). The PBS is aspirated carefully, and 4 ml of 0.05%
trypsin/EDTA (Lonza CC-5012) is added and the flask is returned to
the incubator. The detaching cells can be monitored using the
microscope if desired. As a rule of thumb, fibroblasts should
detach in about 2-3 minutes. Longer exposure could damage the cells
irreversibly. When the cells detach completely, the outside of the
flask is sterilized and brought to the hood. The flask is opened
and 8 ml of Trypsin Neutralizing Solution(CC-5002) [2 ml for each
ml of 0.05% trypsin/EDTA (Lonza CC-5002)] is added. The flask
contents are transferred to a 15 ml conical tube and this tube is
centrifuged at 1000 rpm for 5min. The tube is sterilized with
ethanol and returned to the hood. The supernatant is aspirated,
being careful not to disturb the cell pellet. Then, the pellet is
re-suspended using fresh pre-warmed Lonza FGM-2 medium and the
contents are transferred to the flask/flasks, which were previously
filled with Lonza FGM-2 medium. The flasks are gently agitated to
make sure that the medium covers the entire bottom surface and then
returned to the incubator. Feeding is as indicated above.
Example 2
Embedding Cells In the Dermal Layer
[0218] For embedding fibroblasts into the dermal layer (e.g. gel
matrix), the protocol is as follows. First, the fibroblasts are
detached using the trypsinization protocol described above.
However, the pellet is re-suspended in complete E-medium low
calcium (0.6 mM Ca.sup.++), supplemented with 0.5% (V/V) FBS
(Invitrogen 16140071) and 2% penicillin/streptomycin (invitrogen
15140-122) and then added back to the flasks, where they are
allowed to reach 50-60% confluence. Once again, the fibroblasts are
detached according to the protocol described above. Once
re-suspended, they are embedded into the dermal layer. From Day 0
to Day 1-2, the cells in the dermal layer are fed using complete
E-medium low calcium (0.6 mM Ca.sup.++), supplemented with 0.5%
(V/V) FBS (Invitrogen 16140071) and 100 .mu.m ascorbic acid, RM/TI
transglutaminase 50 .mu.g/ml. From Day 1-2 to Day 3-4, the cells in
the dermal layer are fed using complete E-medium low calcium (1.2
mM Ca.sup.++), supplemented with 0.5% (V/V) FBS (Invitrogen
16140071) and 100 .mu.m ascorbic acid and RM/TI transglutaminase 50
.mu.g/ml. From Day 14-18 on, the cells in the dermal layer are fed
using complete cornification medium (1.8 mM Ca.sup.++),
supplemented with 5% (V/V) FBS (Invitrogen 16140071) and 100 .mu.m
ascorbic acid and RM/TI transglutaminase 50 .mu.g/ml.
Example 3
Preparing the Dermal Layer
[0219] First, pipette tips are cooled by putting into refrigerator
for 15-30 min (Pipettes need to be cold when working with rat-tail
type I collagen in order to avoid coagulation). Both the pipette
tips and the ECM matrix should stay in the ice box during the
procedure.
[0220] In order to calculate the final volume of rat-tail type I
collagen mixture needed, one calculates the number of dermal
equivalent cultures that are needed. This calculation is based on
12 well +3 extra (those are needed to compensate for the ECM matrix
that adheres to the surface of pipette). Where 2.times.10.sup.4
neonatal or adult Human Foreskin Fibroblast per raft are employed
and 12+3 rafts are prepared, one needs 15 X
2.times.10.sup.4=30.times.10.sup.4 fibroblasts (or 300,000
fibroblasts). To impede fibroblasts proliferation, one can
irradiate the fibroblast with 70 Gy.
[0221] Now, to make 150 .mu.raft X (12+3) rafts=2.25 ml. 10% 10X
DMEM or variants *=0.225 ml or 225 .mu.l 10% reconstruction buffer
.sup.+=0.225 ml or 225 .mu.l. 80% ECM matrix=1.8 ml or 1800 .mu.l.
(1.8 ml ECM matrix.times.2.4.times.10 1N NaOH (1M))=43.2 .mu.l 1 M
NaOH (1M) (NaOH makes ECM matrix to coagulate). This is put into
INCUBATOR 37 .degree. C. for 2-4 Hours
[0222] One can trypsinize the fibroblasts using 0.05% trypsin/EDTA
(Corning 25-052 CL) according to protocol described above. One can
then re-suspend the fibroblast pellet in the predetermined amount
of 10X DMEM or variants. This is mixed with the necessary amount of
reconstitution buffer. (Note: best results are obtained when
fibroblasts are collected in active growth phase, which occurs when
fibroblast are between 50 and 70% confluence).
[0223] 100 .mu.l ECM+fibroblast are added to each well and this is
incubated (37.degree. C. for 2 Hours). Thereafter, 100 .mu.l of E
medium is added to the top of each collagen gel. 100 .mu.l of E
medium+RM TG* is then added to the bottom of each collagen gel.
This is incubated (37.degree. C. for 12-16 Hours).
[0224] A variety of collagen containing matrices are contemplated
for making an artificial derma and ECM to embed fibroblasts:
[0225] Tropoelastin : Collagen I : Collagen III : Dermatan sulfate
(1mg:3mg:3mg:0.5mg)
[0226] Col I (3 mg/ml)/Elastin (3mg/ml)
[0227] Col I (3 mg/ml)/Elastin (1mg/ml)
[0228] Col I (10 mg/ml)/MaxGEL
[0229] Col I (3 mg/ml)/Elastin (3mg/ml) 1:1 MaxGel
[0230] Col I (3 mg/ml)/Elastin (3mg/ml)/Col III (3mg/ml) 1:1:1
[0231] MaxGel
[0232] Col I (10mg/ml)/Elastin (10mg/ml)
[0233] Additional embodiments are contemplated:
[0234] 1. A device comprising i) a chamber, said chamber comprising
a lumen, said lumen positioned under ii) a removable top and above
iii) a porous membrane, said membrane positioned above one or more
iv) fluidic channels.
[0235] 2. The device of Claim 1, further comprising a gel
matrix.
[0236] 3. The device of Claim 2, further comprising parenchymal
cells on or in the gel matrix, or both.
[0237] 4. The device of Claim 3, wherein said parenchymal cells are
selected from the group consisting of epithelial cells of the lung
and epithelial cells of the skin.
[0238] 5. The device of Claim 4, wherein said epithelial cells of
the lung are selected from the group consisting of alveolar
epithelial cells and airway epithelial cells.
[0239] 6. The device of Claim 4, wherein said epithelial cells of
the skin comprise keratinocytes.
[0240] 7. The device of Claim 1, further comprising positioned on
the bottom of the membrane so as to be in contact with the fluidic
channels.
[0241] 8. The device of Claim 7, wherein the endothelial cells are
primary cells.
[0242] 9. The device of Claim 8, wherein said primary cells are
small vessel human dermal microvascular endothelial cells.
[0243] 10. The device of Claim 8, wherein said primary cells are
human umbilical vein endothelial cells.
[0244] 11. The device of Claim 8, wherein said primary cells are
bone marrow-derived endothelial progenitor cells.
[0245] 12. The device of Claim 6, wherein said keratinocytes are
epidermal keratinocytes.
[0246] 13. The device of Claim 6, wherein said keratinocytes are
human foreskin keratinocytes.
[0247] 14. The device of Claim 1, wherein said device is a
microfluidic device and said fluidic channels are microfluidic
channels.
[0248] 15. A device comprising i) a chamber, said chamber
comprising a lumen, said lumen comprising ii) a gel matrix, said
gel matrix comprising parenchymal cells, said gel matrix positioned
above iii) a porous membrane, said membrane comprising endothelial
cells in contact with iv) fluidic channels.
[0249] 16. The device of Claim 15, wherein said parenchymal cells
are selected from the group consisting of epithelial cells of the
lung and epithelial cells of the skin.
[0250] 17. The device of Claim 16, wherein said epithelial cells of
the lung are selected from the group consisting of alveolar
epithelial cells and airway epithelial cells.
[0251] 18. The device of Claim 16, wherein said epithelial cells of
the skin comprise keratinocytes.
[0252] 19. The device of Claim 18, further comprising fibroblasts
within the gel matrix, wherein the keratinocytes are on top of the
gel matrix.
[0253] 20. The device of Claim 19, wherein the keratinocytes
comprise more than one layer on top of the gel matrix.
[0254] 21. The device of Claim 15, wherein the endothelial cells
are primary cells.
[0255] 22. The device of Claim 21, wherein said primary cells are
small vessel human dermal microvascular endothelial cells.
[0256] 23. The device of Claim 21, wherein said primary cells are
human umbilical vein endothelial cells.
[0257] 24. The device of Claim 21, wherein said primary cells are
bone marrow-derived endothelial progenitor cells.
[0258] 25. The device of Claim 18, wherein said keratinocytes are
epidermal keratinocytes.
[0259] 26. The device of Claim 18, wherein said keratinocytes are
human foreskin keratinocytes.
[0260] 27. The device of Claim 15, further comprising an open
region in contact with at least one of said gel, said membrane,
said parenchymal cells or said endothelial cells.
[0261] 28. A method of testing a drug, comprising 1) providing a) a
candidate drug and b) device comprising i) a chamber, said chamber
comprising a lumen, said lumen positioned above ii) a porous
membrane, said membrane comprising parenchymal cells and positioned
above one or more iii) fluidic channels; and 2) contacting said
parenchymal cells with said candidate drug.
[0262] 29. The method of Claim 28, wherein said parenchymal cells
are selected from the group consisting of epithelial cells of the
lung and epithelial cells of the skin.
[0263] 30. The method of Claim 29, wherein said epithelial cells of
the lung are selected from the group consisting of alveolar
epithelial cells and airway epithelial cells.
[0264] 31. The method of Claim 29, wherein said epithelial cells of
the skin comprise keratinocytes.
[0265] 32. The method of Claim 31, further comprising fibroblasts
within the gel matrix, wherein the keratinocytes are on top of the
gel matrix.
[0266] 33. The method of Claim 28, wherein said chamber lacks a
covering and said candidate drug is introduced into said lumen
under conditions such that said parenchymal cells are
contacted.
[0267] 34. The method of Claim 28, wherein said candidate drug is
in an aerosol.
[0268] 35. The method of Claim 28, wherein said candidate drug is
in a paste.
[0269] 36. The method of Claim 28, wherein said device further
comprises a removable top and said method further comprises, prior
to step 2), removing said removable top.
[0270] 37. A method of testing an agent comprising 1) providing a)
an agent and b) microfluidic device comprising i) a chamber, said
chamber comprising a lumen, said lumen comprising ii) a gel matrix
comprising cells in, on or under said gel matrix, said gel matrix
positioned above iii) a porous membrane and under iv) a removable
cover, said membrane positioned above one or more v) fluidic
channels; 2) removing said removable cover; and 3) contacting said
cells in, on or under said gel matrix with said agent.
[0271] 38. The method of Claim 37, wherein said agent is in an
aerosol.
[0272] 39. The method of Claim 37, wherein said agent is in a
paste.
[0273] 40. The method of Claim 37, wherein said agent is in a
liquid, gas, gel, semi-solid, solid, or particulate form.
[0274] 41. A device comprising i) a chamber, said chamber
comprising a lumen and projections into the lumen, said lumen
comprising ii) a gel matrix anchored by said projections, said gel
matrix positioned above iii) a porous membrane, said membrane
positioned above one or more iv) fluidic channels.
[0275] 42. The device of Claim 41, wherein fibroblasts are within
the gel matrix and keratinocytes are on top of the gel matrix.
[0276] 43. The device of Claim 42, wherein the keratinocytes
comprise more than one layer on top of the gel matrix.
[0277] 44. The device of Claim 41, wherein a layer of endothelial
cells is positioned on the bottom of the membrane so as to be in
contact with the fluidic channels.
[0278] 45. The device of Claim 44, wherein the endothelial cells
are primary cells.
[0279] 46. The device of Claim 45, wherein said primary cells are
small vessel human dermal microvascular endothelial cells.
[0280] 47. The device of Claim 45, wherein said primary cells are
human umbilical vein endothelial cells.
[0281] 48. The device of Claim 45, wherein said primary cells are
bone marrow-derived endothelial progenitor cells.
[0282] 49. The device of Claim 42, wherein said keratinocytes are
epidermal keratinocytes.
[0283] 50. The device of Claim 42, wherein said keratinocytes are
human foreskin keratinocytes.
[0284] 51. The device of Claim 41, further comprising a removable
cover.
[0285] 52. The device of Claim 41, wherein said device is a
microfluidic device and said fluidic channels are microfluidic
channels.
[0286] 53. A microfluidic device comprising i) a chamber, said
chamber comprising a lumen and projections into the lumen, said
lumen comprising ii) a gel matrix anchored by said projections,
said gel matrix comprising fibroblasts and keratinocytes, said gel
matrix positioned above iii) a porous membrane, said membrane
comprising endothelial cells in contact with iv) microfluidic
channels.
[0287] 54. The device of Claim 53, wherein the membrane is above
said fluidic channels and wherein the layer of endothelial cells is
positioned on the bottom of the membrane so as to be in contact
with the fluidic channels.
[0288] 55. The device of Claim 53, wherein the fibroblasts are
within the gel matrix and the keratinocytes are on top of the gel
matrix.
[0289] 56. The device of Claim 55, wherein the keratinocytes
comprise more than one layer on top of the gel matrix.
[0290] 57. The device of Claim 53, wherein the endothelial cells
are primary cells.
[0291] 58. The device of Claim 57, wherein said primary cells are
small vessel human dermal microvascular endothelial cells.
[0292] 59. The device of Claim 57, wherein said primary cells are
human umbilical vein endothelial cells.
[0293] 60. The device of Claim 57, wherein said primary cells are
bone marrow-derived endothelial progenitor cells.
[0294] 61. The device of Claim 53, wherein said keratinocytes are
epidermal keratinocytes.
[0295] 62. The device of Claim 53, wherein said keratinocytes are
human foreskin keratinocytes.
[0296] 63. The device of Claim 53, wherein said matrix comprises
collagen.
[0297] 64. The device of Claim 53, wherein said collagen matrix is
between 0.2 and 6 mm in thickness.
[0298] 65. A method of testing a drug on keratinocytes, comprising
1) providing a) a candidate drug and b) microfluidic device
comprising i) a chamber, said chamber comprising a lumen and
projections into the lumen, said lumen comprising ii) a gel matrix
anchored by said projections, said gel matrix comprising
fibroblasts and keratinocytes, said gel matrix positioned above
iii) a porous membrane, said membrane comprising endothelial cells
in contact with iv) fluidic channels; and 2) contacting said
keratinocytes with said candidate drug.
[0299] 66. The method of Claim 28, wherein the fibroblasts are
within the gel matrix and the keratinocytes are on top of the gel
matrix.
[0300] 67. The method of Claim 28, wherein said chamber lacks a
covering and said candidate drug is introduced into said lumen
under conditions such that said keratinocytes are contacted.
[0301] 68. The method of Claim 28, wherein said candidate drug is
in an aerosol.
[0302] 69. The method of Claim 28, wherein said candidate drug is
in a paste.
[0303] 70. The method of Claim 28, wherein said microfluidic device
further comprises a removable top and said method further
comprises, prior to step 2), removing said removable top.
[0304] 71. The method of Claim 28, wherein said microfluidic device
further comprises an open region in contact with at least one of
said gel matrix, said membrane, said keratinocytes or said
endothelial cells.
[0305] 72. A method of testing an agent comprising 1) providing a)
an agent and b) microfluidic device comprising i) a chamber, said
chamber comprising a lumen and projections into the lumen, said
lumen comprising ii) a gel matrix anchored by said projections and
comprising cells in, on or under said gel matrix, said gel matrix
positioned above iii) a porous membrane and under iv) a removable
cover, said membrane positioned above one or more v) fluidic
channels; 2) removing said removable cover; and 3) contacting said
cells in, on or under said gel matrix with said agent.
[0306] 73. The method of Claim 72, wherein said agent is in an
aerosol.
[0307] 74. The method of Claim 72, wherein said agent is in a
paste.
[0308] 75. The method of Claim 72, wherein said agent is in a
liquid, gas, gel, semi-solid, solid, or particulate form.
Still Additional Embodiments are Contemplated:
[0309] 28. A fluidic cover comprising a fluidic channel, said
fluidic cover configured to engage a microfluidic device.
[0310] 29. The fluidic cover of Claim 28, wherein said microfluidic
device comprises an open chamber, and wherein said fluidic cover
configured to cover and close said open chamber.
[0311] 30. The fluidic cover of Claim 28, further comprising one or
more electrodes.
[0312] 31. An assembly comprising a fluidic cover comprising a
fluidic channel, said fluidic cover detachably engaged with a
microfluidic device.
[0313] 32. The assembly of Claim 31, wherein said microfluidic
device comprises an open chamber, and wherein said fluidic cover
configured to cover and close said open chamber.
[0314] 33. The assembly of Claim 32, wherein said open chamber
comprises a non-linear lumen.
[0315] 34. The assembly of Claim 33, wherein said non-linear lumen
is circular.
[0316] 35. The assembly of Claim 31, wherein said fluidic cover
further comprises one or more electrodes.
[0317] 36. A method of making an assembly, comprising: a) providing
a fluidic cover comprising a fluidic channel, said fluidic cover
configured to engage b) a microfluidic device, said microfluidic
device comprises an open chamber, and wherein said fluidic cover
configured to cover and close said open chamber; and b) detachably
engaging said microfluidic device with said fluidic cover so as to
make an assembly.
[0318] 37. The method of making an assembly of Claim 36, wherein
said open chamber comprises a non-linear lumen.
[0319] 38. The method of making an assembly of Claim 37, wherein
said non-linear lumen is circular.
[0320] 39. The method of making an assembly of Claim 36, wherein
said fluidic cover further comprises one or more electrodes.
* * * * *